CN110187119B - Methods and compositions for the detection and treatment of preeclampsia - Google Patents
Methods and compositions for the detection and treatment of preeclampsia Download PDFInfo
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- CN110187119B CN110187119B CN201910197163.6A CN201910197163A CN110187119B CN 110187119 B CN110187119 B CN 110187119B CN 201910197163 A CN201910197163 A CN 201910197163A CN 110187119 B CN110187119 B CN 110187119B
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Abstract
Methods, kits and compounds related to the diagnosis, treatment and/or prevention of preeclampsia are provided.
Description
The application is a divisional application of a Chinese patent application with application numbers of 200980153533.0, 2009-11/2 and an invention name of a method and a composition for detecting and treating preeclampsia, and is a national phase application with international application numbers of PCT/US2009/005957, wherein the international application requires priority of US provisional applications with application numbers of 61/197,914 and 61/206,534 respectively, and the application numbers of PCT/US2009/005957 are respectively 10/31 and 2009-1/29.
Related patent
This patent application claims the following two priorities: U.S. provisional patent No. 61/197,914, filed on 31/10/2008, entitled "preeclampsia is a disease characterized by specific supramolecular polymerization of misfolded proteins"; U.S. provisional patent No. 61/206,534, filed 1/29 of 2009, entitled "preeclampsia is a disease characterized by specific supramolecular polymerization of misfolded proteins, detectable using antibodies that recognize specific protein oligomer conformations," both of which are incorporated herein by reference in their entirety.
Background
In the United states, 6-8% of pregnant women are afflicted with preeclampsia (Hauth, J.C. et al., Obstet Gynecol.95(1):24-8,2000), with a morbidity of 23.6 out of every 1000 deliveries (Samadi, A.R. et al., Obstet Gynecol.87(4):557-63, 1996). Preeclampsia is believed to be responsible for 20% of maternal related female deaths (MacKay, A.P.et., Paediata woman epidemibiol.19 (3): 206;. 14,2005), which is the main cause of therapeutic preterm delivery (MIPTD) (Fronterhouse, W.et., J Matern Fetal Med.10(3): 162;. 5,2001.), which should be responsible for 10% of all preterm deliveries (Fronterhouse, W.et., J Matern Fetal Med.10(3): 162;. 5, 2001.). Preeclampsia (PE) is characterized by the onset of elevated blood pressure and proteinuria at 20 weeks after pregnancy (ACOG Committee on Practice bulletins. Obstet Gynecol.99(1): 159. 67, 2002.). Severe preeclampsia (sPE) is considered if blood pressure and proteinuria continue to increase or symptoms of end organ damage, including fetal growth restriction, occur. The course of severe preeclampsia is accompanied by progressive deterioration of the physical condition of women, and iatrogenic labor remains the only definitive treatment. Measures that may be taken by a care physician include weighing the risk of immediate delivery to an immature fetus versus the risk of both mother and child suffering preeclampsia. In this regard, the current approach is to closely monitor the status of the mother and fetus and to manually produce it as the final treatment. (Zamorski, M.A. & Green, L.A.am Fam Physician 64:263-70,216,2001.)
Today, there is no clinical test that can predict or diagnose pre-eclampsia or how severe a particular patient is expected to develop. Early symptoms include persistent headache, blurred vision or sensitivity to light, and abdominal pain. However, preeclampsia is usually not diagnosed until elevated blood pressure and protein in the urine (proteinuria) are found, which are typically found in routine care examinations twenty weeks after pregnancy (Roberts JM, Cooper DW. Pathogenesis and genetics of pre-eclampsia. Lancet.2001; 357(9249): 53-56). Severe symptoms of preeclampsia after such diagnosis, including epilepsy, cerebral hemorrhage, disseminated intravascular coagulation and renal failure, may occur rapidly. These methods are inaccurate and provide little information on the likelihood of most severe symptoms developing. Moreover, current diagnostic methods require physician supervision and invasive procedures, further delaying and complicating early and rapid diagnosis. There is therefore an urgent need to develop a more early and accurate method for detecting and diagnosing preeclampsia and the associated proteinuria and hypertension disorders without the need for supervision by a physician.
Disclosure of Invention
As described below, applicants have demonstrated that Preeclampsia (PE) is a condition that is limited to pregnant women characterized by protein misfolding (formation of abnormal or misfolded protein aggregates). In addition, they demonstrated that very characteristic abnormal protein aggregates (we also refer to misfolded protein aggregates or intermediates) appear in urine and accumulate in the placenta of pregnant women (patients) who have acquired preeclampsia and are congophilic. These abnormal protein aggregates are associated with the presence of preeclampsia, and their presence in urine and/or placental tissue also indicates the presence of preeclampsia. In the present specification, the term "supramolecular aggregates of misfolded proteins" (referred to as "supramolecular aggregates") is intended to include both soluble and insoluble protein aggregates. The supramolecular aggregates of misfolded proteins described herein that accompany the development of preeclampsia may be used to determine the development of preeclampsia in a pregnant woman, as well as the likelihood or risk of preeclampsia in a pregnant woman.
Recent proteomic findings have made it possible to find biomarker molecules and consider preeclampsia as a new view of misfolding disorders. As described herein, applicants have determined the occurrence and nature and level of protein misfolding in PE. They have demonstrated that PE is characterized by an increased excretion of misfolded proteins, a phenomenon that can be detected using the affinity of misfolded protein aggregates for specific dyes, such as the self-assembling azo dye Congo Red (CR).
Protein conformation disorders such as senile dementia, light chain amyloidosis and raney virus disease are formed by the formation and aggregation of starch-containing fibers due to the defensive folding of intracellular proteins into abnormal 3D structures. Applicants have discovered that soluble pre-amyloid oligomers (intermediates in fiber assembly, i.e., misfolded protein intermediates) have protein toxic effects that can lead to endothelial damage and oxidative stress, a process that plays a pathogenic role in the severe pre-eclampsia (sPE). Applicants have found that the detection of the presence of soluble pro-amyloid oligomers may indicate or be predictive of severe PE (severe PE) and pre-severe PE (pre-severe PE) (pre-preeclampsia, i.e. a subclinical state prior to sPE which may become clinically dominant).
Thus, applicants have discovered and identified the nature of soluble pro-amyloid oligomers in urine in such conditions characteristic of pregnant women. Their efforts were to first discover conformational disorders in the world where PE is characterized by the amyloid-like protein assembly of proteins.
Further work described herein using antibodies that can recognize proteins and protein oligomers with unique folded conformations supports the discovery that misfolded protein intermediates tend to assemble into porous structures (amyloid channels) that may play a role in the manifestation of clinical disease. Abnormal and/or excessively misfolded protein aggregates accumulate in the urine and/or placenta of patients who have been diagnosed with PE or are destined to be PE, suggesting that these abnormal protein aggregates are causative factors in the disease pathology. This new finding provides the basis for new diagnostic and therapeutic strategies for the treatment (partial or total reduction in the onset or progression, reversal of the disease) of preeclampsia. For example, the use of immunological or pharmacological means to prevent the formation of such structures, to reverse (interfere with, disassemble) the already existing supramolecular aggregates provides a new approach to therapeutic intervention in the treatment of preeclampsia.
Such abnormal protein aggregates (supramolecular amyloidogenic protein assembly of proteins) are also described herein, in addition to their affinity for congo red. One component of supramolecular aggregates found in urine and placental tissue of preeclamptic patients is serpin a1(alpha-1 antitrypsin) or a polypeptide fragment of serpin a 1. Thus, preeclampsia has a similar characteristic to other diseases (e.g., alpha-1 antitrypsin deficiency): in alpha-1 antitrypsin deficiency, accumulation of misfolded alpha-1 antitrypsin leads to damage of hepatocytes and cirrhosis of the liver (n.Engl.1ed.346:45-53 (2002); J.Clin.Inv.110:1585-1590 (2002)). In addition, ceruloplasmin (ceruloplasmin), heavy-chain IgG (heavy-chain IgG) and light-chain IgG (light-chain IgG), interferon inducible protein 6-16(interferon-inducible protein 6-16) (IFI6-16, G1P3) and fragments thereof were identified as components of misfolded protein aggregates in Congo-red proteinuria of PE. In certain embodiments described herein and described below, are isolated abnormal protein aggregates that accompany pre-eclampsia and that appear in urine and placental tissue, and that contain at least one (or more) of the following components: serpin A1, ceruloplasmin, heavy chain IgG, light chain IgG, interferon inducible protein 6-16 and fragments of each.
One of the embodiments of the invention described herein is a non-invasive urine diagnostic and prognostic test or assay based on detecting the presence or absence (qualitative) or amount (quantitative) of the above-described abnormal protein aggregates that exhibit affinity for congo red (congo red-philic) with preeclampsia during pregnancy and is a measure of the integrity of protein misfolding. The use of Congo Red Retention (CRR) to assess the overall protein misfolding load (global protein misfolding load) is a simple diagnostic test to diagnose PE and indicated delivery (IND), which is a large factor in preterm labor. Described herein are diagnostic and prognostic assays, in some embodiments, specifically based on evaluating or analyzing a sample (e.g., urine or placental tissue) obtained from a pregnant woman to detect whether the sample contains aggregates of congo red-philic abnormal proteins associated with preeclampsia. However, the reader should understand that: such diagnostic or prognostic assays may employ a wide variety of reagents that are capable of detecting or determining the presence or amount of abnormal protein aggregates associated with preeclampsia in a sample, congo red-philic being only one of the descriptive characteristics of misfolded protein aggregates. Other features include: such as the composition and conformation of abnormal protein aggregates, which in turn define other properties of misfolded protein aggregates, such as their size, interaction with other dyes, and binding to antibodies, which may be used in the diagnostic and prognostic methods described above.
Described herein is a method for diagnosing or aiding in the diagnosis of preeclampsia in a pregnant woman by detecting supramolecular aggregates (abnormal protein aggregates, misfolded protein intermediates, supramolecular amyloid protein assembly of proteins) of one (or more) misfolded protein that appear in association with preeclampsia in the urine or placental tissue of the pregnant woman. It should be understood that the detection assays described herein may be performed at any time during the pregnancy. In some particular embodiments, the diagnostic tests described herein may be performed on women who are scheduled to become pregnant.
Such supramolecular aggregates can be detected in urine or placental tissue using a range of existing techniques and methods known in the art. One suitable technique is to detect abnormal protein aggregates in urine or placental tissue at levels associated with preeclampsia. Such techniques may utilize dyes, small molecules, fluorescent compounds, or other agents (e.g., an antibody, antibody fragment, antibody peptidomimetic) that bind to or interact with the abnormal protein aggregate to label, visualize, or otherwise alter the aggregate so that it can be detected. In this and other embodiments, one result is that the supramolecular aggregate/abnormal protein aggregate and urine or placental tissue samples differ from the urine or placental tissue and components thereof originally obtained (i.e., the samples have been modified, e.g., by addition of reagents to analyze them, by changing temperature or dilution or concentration; the abnormal protein aggregate has been labeled, displayed or changed so that they can be detected). It will be appreciated that when a dye is used, the dye that can be used to detect supramolecular aggregation as described herein may have one or more of the following characteristics: a self-assembling dye, a non-self-assembling dye, a heteroaromatic dye, an azo dye, a fiber-specific dye and/or other dyes. These dyes may include, but are not limited to: congo red, curcumin analogs; x-34(1, 4-bis (3-carboxy-4-hydroxyphenylvinyl) benzene) (1,4-bis (3-methoxy-4-hydroxyphenylethyl) -benzene); thioflavin s (thioflavin s); thioflavin t (thioflavin t); nile Red (Nile Red); acridine orange (acridine orange); naphthalene-8-amino-sulfonate (ANS); Bis-ANS; 4- (dicyanovinyl) julolidine (4- (dicyanovinyl) -juliolidine) (DCVJ); AO1987 (oxazine dyes); fluorescent styryl dyes (fluorescent styryl dyes); BF-168: (6-2-Fluoroethoxy) -2- [2- (4-methylaminophenol) vinyl ] benzoxazole ((6-2-Fluoroethoxy) -2- [2- (4-methylenedianil) ethyl ] benzoxazole); BSB: (trans ) -1-bromo-2, 5-bis- (3-hydroxycarbonyl-4-hydroxy) styrylbenzene ((trans, trans) -l-bromo-2,5-bis- (3-hydroxycarbonyl-4-hydroxy) styrylbenzene); quinolinehydrazone compounds (e.g., 4-methyl-7-methoxy-2- (4-quinolinylmethylenehydrazine) quinoline) (i.e., 4-methyl-7-methoxy-2- (4-quinolinylmethylenehydrazino) quinoline), aurantiamide G (i.e., Chrysamine-G); rifampin (rifampicin); melatonin; baicalein (baicalein); scyllitol (e.g., scyllitol (Cocositol), Quercinitol, 1,3,5/2,4, 6-hexahydroxycyclohexane) and derivatives thereof; imaging probes, such as [11C ] -PIB: n-methyl [11C ]2- (4 '-methylaminophenol-6-hydroxybenzotriazole) (N-methyl [11C ]2- (4' -methylenephenyl-6-hydroxybenzothiazole)); stilbene benzotriazole (stilbenylbenzothiazole) and derivatives thereof.
Self-assembling dyes include, for example, congo red, Evans Blue (Evans Blue), bis-azo ANS, and non-self-assembling dyes include, for example, ANS and Trypan Blue (Trypan Blue).
As described herein, these abnormal protein aggregates show affinity for the self-assembling azo dye congo red, so their presence in urine or placental tissue can be determined (qualitatively and/or quantitatively) by detecting the congo red-philic property in urine or placental tissue from a pregnant woman to be detected (tested) for preeclampsia. Described herein is a method of diagnosing or aiding in the diagnosis of preeclampsia in a pregnant woman, detecting the congophilic properties of urine, such as by dot blot attachment and spectral shift analysis, indicating that a pregnant woman has preeclampsia (indicating that the pregnant woman has preeclampsia) or will have preeclampsia (further testing can be performed using known methods to determine if desired). As described herein, although we refer to these abnormal proteins as congophilic, this is a descriptive term, the reader should understand that these abnormal proteins can be detected by a number of other means, including other agents, such as other dyes (e.g., other self-assembling dyes), thioflavins (thioflavins S, thioflavins T) and antibodies (e.g., polyclonal, such as A11 antibody, Officer antibody, OC antibody, and monoclonal, such as M118, M204, M205, M89 (e.g., M89-17) and M09) or by observing the aggregates using, for example, polarized light microscopy, fluorescence microscopy or electron microscopy.
The abnormal protein aggregates or fragments detected by detection of congophilic properties are the abnormal protein aggregates described herein, which contain at least one (one or more) of serpin A1, ceruloplasmin, heavy chain IgG, light chain IgG, interferon inducible proteins 6-16, and fragments of each.
It will be understood that in all of the examples described herein, various antibodies (polyclonal or monoclonal) may be used. In particular examples, at least one (one or more) of the following antibodies or equivalents thereof (antibodies that recognize (bind) in nearly the same manner) may be used, such as for the same epitope or conformation: a11 antibody, Officer antibody, OC antibody, M118 antibody, M204 antibody, M205 antibody, M89 antibody (e.g., M89-17) and M09 antibody.
In certain embodiments, a method of diagnosing or aiding in the diagnosis of preeclampsia in a pregnant woman comprises: (a) obtaining a urine sample or a placental tissue sample from a pregnant woman; (b) combining (contacting) the sample obtained in (a) with an agent (e.g., a dye such as congo red, or an antibody) that binds to or labels abnormal protein aggregates associated with PE or components thereof under conditions that allow the agent to bind to or interact with the abnormal protein aggregates, thereby producing (labeled) abnormal protein aggregates or components thereof (e.g., detectable serpina-2 or other components) that can be detected and (c) determining whether binding or other interaction occurs in (a), e.g., by determining whether detectable abnormal protein aggregates or components are present in the sample, such that binding indicates that the woman is suffering from preeclampsia, the woman may be diagnosed as having or about to suffer from preeclampsia. Her status (whether or not it has preeclampsia) can be further examined by known means (as described herein) to confirm the results obtained by the current methods. The agent that binds to or displays or labels the abnormal protein may be of a variety of classes, such as a dye (e.g., congo red, thioflavin S, thioflavin T, evans blue, trypan blue, ANS, bis-azo ANS), a radioactive tracer (e.g., (11) C pittsburgh compound b (pib)) or an antibody against (binding to) the abnormal protein aggregate, such as an antibody that binds to a characteristic region or conformational feature of the abnormal protein aggregate, or to a characteristic region or feature of one or more of its components (e.g., one or more of serpin a1, ceruloplasmin, heavy chain IgG, light chain IgG, interferon 6-16 or fragments of each).
In a specific embodiment, a method of diagnosing or aiding in diagnosing a pregnant woman suffering from preeclampsia comprises: (a) obtaining a urine sample or a placental tissue sample from a pregnant female; (b) mixing the sample obtained in (a) with a dye (e.g., congo red) capable of staining at least one protein or protein fragment present in abnormal protein aggregates under conditions wherein the dye stains the protein in the sample, thereby producing a urine sample or a placental tissue sample comprising the dye; and (c) analyzing the urine sample or placental tissue sample obtained in (b) to determine (confirm whether the sample contains) the presence of supramolecular aggregates (abnormal protein aggregates) that are associated with preeclampsia and staining with the dye, wherein if the sample contains supramolecular aggregates that are associated with preeclampsia and stained with the dye, the staining indicates that the woman has preeclampsia, the woman is diagnosed with or is likely to have preeclampsia. If the sample contains such stained misfolded protein aggregates, she is more likely to have preeclampsia than if the sample does not contain dye-stained abnormal protein aggregates that accompany preeclampsia. The presence of dye-stained abnormal protein aggregates that accompany preeclampsia is indicative that the woman has or is likely to have preeclampsia. Her status (whether or not it has preeclampsia) can be further examined by known means (as described herein) to confirm the results obtained by the current methods. The abnormal protein aggregates detected are those described herein, which contain at least one or more of serpin A1, ceruloplasmin, heavy chain IgG, light chain IgG, interferon inducible protein 6-16, and fragments of each. Detection of stained abnormal protein aggregates can be accomplished by known methods such as dot blot analysis or by merely visually observing an area on a stained support surface (e.g., filter paper) comprising a combination of a urine sample (or placental tissue sample) and a dye (e.g., a mixture of a urine sample and congo red).
A pregnant woman determined to have, be likely to have, or be at risk of developing preeclampsia using the embodiments described herein may be further evaluated for such or confirmed the presence/risk of preeclampsia by performing an additional test, such as the methods described previously or by one or more methods known to those of skill in the art (i.e., blood pressure measurement, edema assessment, abdominal pain, the occurrence of headache, and visual problems).
The detection of congo red-philic abnormal protein aggregates in urine described herein may be used not only to diagnose pre-eclampsia that is already present, but also to predict the future occurrence of pre-eclampsia in pregnant women, and may also be considered to predict an increased likelihood that a pregnant woman will develop pre-eclampsia. In one embodiment, the method of predicting or aiding in predicting that a pregnant woman will develop (will develop) preeclampsia comprises obtaining a urine or placental tissue sample from the pregnant woman and analyzing the urine or placental tissue sample for the presence of abnormal protein aggregates associated with preeclampsia (identifying the presence of abnormal protein aggregates in the sample), the sample being more likely to contain abnormal protein aggregates associated with preeclampsia than is the case in a normal human being. The reader should understand that: the methods of occurrence and/or risk of occurrence of PE described herein include methods of detecting the presence/absence of abnormal protein aggregates that accompany preeclampsia (for rapid qualitative analysis) and methods of quantifying the amount of supramolecular aggregates detected in a sample and/or their precise nature (i.e., identifying the protein in the aggregates, identifying the conformation of the aggregates, etc.).
The abnormal protein aggregate detected may be a supramolecular aggregate(s) as described herein, comprising at least one (one or more) of the following components: SerpinA1, ceruloplasmin, heavy chain IgG, light chain IgG, interferon inducible protein 6-16 or fragments of each of these proteins. Methods of predicting whether a pregnant woman will develop preeclampsia can be accomplished, for example, (a) obtaining a urine sample or placental tissue sample from the pregnant woman; (b) combining the urine sample or placental tissue sample with a reagent that binds to or interacts with misfolded protein aggregates that occur in association with preeclampsia under conditions that allow the reagent to bind to or interact with such misfolded protein aggregates or components thereof, thereby producing a composition or component thereof (e.g., detectable serpin a1 or other component) comprising detectable (labeled) abnormal protein aggregates; and (c) determining whether the binding or other interaction of (b) has occurred, e.g., by determining whether detectable misfolded protein aggregates or components thereof are present in the sample, if any, such that the pregnant woman is more likely to develop preeclampsia during pregnancy (and is more likely to develop preeclampsia) than if detectable protein aggregates were not present in the sample. The agent that binds to or displays or labels the abnormal protein may be one of several types, such as a dye (e.g. congo red, thioflavin S, thioflavin T, evans blue, trypan blue, ANS, bis-azo ANS), a radiotracer (e.g. (11) C pittsburgh complex b (pib)) or an antibody against (binding to) the abnormal protein aggregate, such as an antibody that can bind to a characteristic region or specific conformation or one or more aggregate components, such as one or more serpin a1, ceruloplasmin, heavy chain IgG, light chain IgG, interferon inducible protein 6-16 and one or more fragments of these proteins.
In a particular embodiment, a method of predicting or aiding in predicting the likelihood that a pregnant woman will develop (will develop) preeclampsia comprises: (a) obtaining a urine sample or a placental tissue sample from a pregnant woman; (b) mixing the sample with a dye (e.g., congo red) capable of staining proteins or protein fragments in supramolecular aggregates that accompany preeclampsia under conditions that allow the dye to stain proteins in the sample, thereby producing a sample containing the dye; and (c) analyzing the urine sample or placental tissue sample obtained in (b) to determine (confirm whether the sample contains) the presence of dye-stained supramolecular aggregates that accompany preeclampsia, and if the sample contains dye-stained supramolecular aggregates that accompany preeclampsia, she is more likely (will be more likely) to develop preeclampsia than if the sample does not contain dye-stained abnormal protein aggregates that accompany preeclampsia. Stained aggregates can be detected by using known methods such as dot blot analysis or by merely visually observing an area on a stained support surface (e.g., filter paper) containing a combination of a urine sample (or placenta tissue sample) and a dye (e.g., a mixture of a urine sample and congo red).
Similarly, the risk that a pregnant woman will develop preeclampsia can be assessed by the methods described herein. In one embodiment, a method for determining the risk that a pregnant woman will have (will develop) preeclampsia includes obtaining a urine or placental tissue sample from the pregnant woman and analyzing the urine or placental tissue sample for the presence of misfolded protein aggregates that occur with preeclampsia (identifying the presence of misfolded protein aggregates in the sample), the woman being at greater risk if the sample contains misfolded protein aggregates that occur with preeclampsia than if the sample does not contain misfolded protein aggregates that occur with preeclampsia. In a specific embodiment, a method of assessing and determining the risk that a pregnant woman will develop preeclampsia comprises (a) obtaining a urine sample or placental tissue sample from the pregnant woman; (b) combining the urine sample or placental tissue sample with an agent that binds to or interacts with misfolded protein aggregates that accompany preeclampsia under conditions that allow the agent to bind to or interact with such misfolded protein aggregates or components thereof, thereby producing detectable (labeled) protein aggregates or components thereof (e.g., detectable Serpin-2 or other components); and (c) determining whether the binding or other interaction of (b) has occurred, e.g., by determining whether detectable misfolded protein aggregates or components thereof are present in the sample, if present, the pregnant woman being at greater risk of developing preeclampsia than the individual in the sample for whom no detectable protein aggregates are present. The agent that binds to or displays or labels the abnormal protein may be one of several types, such as a dye (e.g. congo red, thioflavin S, thioflavin T, evans blue, trypan blue, ANS, bis-azo ANS), a radiotracer (e.g. (11) C pittsburgh complex b (pib)) or an antibody against (binding to) the abnormal protein aggregate, such as an antibody that can bind to a characteristic region or specific conformation or one or more aggregate components, such as one or more serpin a1, ceruloplasmin, heavy chain IgG, light chain IgG, interferon inducible protein 6-16 and one or more fragments of these proteins.
In a specific embodiment, a method of assessing or estimating that a pregnant woman will have (will develop) preeclampsia comprises: (a) obtaining a urine sample or a placental tissue sample from a pregnant woman; (b) mixing the sample with a dye (e.g., congo red) that stains at least one protein or protein fragment of a misfolded protein aggregate that occurs in connection with preeclampsia under conditions that allow the dye to stain proteins in the sample, thereby producing a sample containing the dye; and (c) analyzing the urine sample or placental tissue sample obtained in (b) to determine (confirm whether the sample contains) the presence of dye-stained misfolded protein aggregates that accompany pre-eclampsia, wherein if the sample contains dye-stained misfolded protein aggregates that accompany pre-eclampsia, then the pregnant woman is at greater risk of developing (more likely to develop) pre-eclampsia than if the sample does not contain dye-stained misfolded protein aggregates that accompany pre-eclampsia. Detection of stained aggregates can be accomplished by using known methods such as dot blot analysis or simply observing the area (on a surface, such as filter paper) of the stained surface containing the dye-bound urine or placenta tissue sample (e.g., a mixture of urine and congo red).
Placental tissue samples stained with congo red also showed characteristic properties of such protein aggregates and detectable staining (e.g., congo red staining). Other markers of supramolecular aggregates in the placenta may also be used to diagnose or aid in the diagnosis of preeclampsia in pregnant women; predicting or aiding in predicting the likelihood that a pregnant woman will have (will develop) preeclampsia; assessing and estimating the risk that a pregnant woman will have (will develop) preeclampsia.
In another embodiment, the method is a method of diagnosing or aiding in the diagnosis of preeclampsia in a pregnant woman, i.e., the detection (determination of the presence or absence) of supramolecular aggregates that appear with preeclampsia in urine or placental tissue obtained from the pregnant woman, by using antibodies that recognize (bind to) proteins and protein oligomers described herein that have formed a unique folded conformation with preeclampsia or antibodies that recognize (bind to) supramolecular aggregates that appear with preeclampsia and/or a protein or polypeptide component of such aggregates as a component of such an aggregate. The presence of such supramolecular aggregates is predictive (predictive of the presence of the woman in question) of preeclampsia.
Also described herein is an in vitro method of diagnosing whether a subject (a pregnant woman) is suffering from preeclampsia or predicting whether preeclampsia is likely to be expected in the future, comprising the step of detecting abnormal/misfolded protein aggregates (supramolecular aggregates) in a urine or placenta sample from the subject. In one embodiment, supramolecular aggregates (abnormal/misfolded protein aggregates) are detected in a urine sample. In another embodiment, abnormal/misfolded protein aggregates are detected in a placental sample. In any of the above embodiments, abnormal protein aggregates may be detected by congo red staining, such as by dot blot attachment and/or spectral shift analysis, or by thioflavin S staining. In any embodiment, the aberrantly/misfolded protein aggregate may comprise serpin A1(alpha-1 antitrypsin) and/or a polypeptide fragment and/or a ceruloplasmin fragment and/or a heavy chain IgG and/or a light chain IgG.
Also described herein is a protein polymerization inhibitor for use in a method of treating and/or preventing a pregnant woman from developing preeclampsia. Protein aggregation inhibitors for use in a method of treating and/or preventing preeclampsia in a pregnant woman comprising 1) a protein aggregation inhibitor that inhibits the reformation of supramolecular aggregates; 2) protein inhibitors that reverse the supramolecular aggregates that were present before; 3) protein inhibitors that inhibit the reformation of supramolecular protein aggregates and reverse or disassemble supramolecular protein aggregates that were present before; 4) inhibitors of protein aggregates that stabilize the original conformation of the protein, thus reducing the rate of aberrant folding and subsequent polymerization; 5)1) to 4) that inhibits aggregation of serpin A1(alpha-1 antitrypsin) and/or polypeptide fragments; 1) any one of the protein aggregation inhibitors of (1) to (4), which inhibits aggregation of serpin A1(alpha-1 antitrypsin) and/or a polypeptide fragment and is selected from the group consisting of: (a) trimethylamine N-oxide (trimethylamine N-oxide); (b) trimethylamine N-oxide related compounds; (c) (ii) a fliaiig peptide; and (d) polypeptides and derivatives related to the FLEAIG peptide; 6)1) to 5), and is selected from the group consisting of: (a) trimethylamine N-oxide; (b) trimethylamine N-oxide related compounds; (c) (ii) a fliaiig peptide; and (d) polypeptides and derivatives related to the FLEAIG peptide; 7)1) to 5) and is a trans-beta amyloid agent; 8) a polypeptide inhibitor of 7), and the anti-beta-amyloid agent is selected from: p-aminophenol, 2-Amino-4-Chlorophenol pentapeptide iA β 5(LPFFD) (2-Amino-4-Chlorophenol pentapeptide iAP5) (LPFFD) (Axonyx corporation); the a β aggregation inhibitor PPI-1019 (Praecis); aminopolysaccharide (GAG) peptidomimetic NC-531 (neurohem); the antibiotics Clioquinol (Clioquinol) (Prana Biotech Co., Ltd.), the small molecule cyclodextrins, the natural products from the Ginkgo biloba extract EGb761 ((Dr. Willman Schwabe GmbH & Co.; polyphenols; SP-233, a 22R-hydroxycholesterol derivative (Samaritan pharmacy), Apomorphine (Apomorphine), and A.beta.immunization (Elan pharmaceuticals, Acumen's ADDL technology.) see Drug Discovery Today: Therapeutic Stretageties 2004Elsevier Ltd 1(1): 7-12.
Described herein is a method for studying and testing therapies (i.e., drugs) that can be used to treat preeclampsia. For example, drugs, including those known or later discovered, that reduce the severity of other misfolding disorders may be detected. For example, those agents that have been evaluated for effectiveness in treating conditions such as Alzheimer's disease, light chain amyloidosis, and Raney virus disease may be used to evaluate effectiveness in treating preeclampsia, including preventing its onset, reducing the extent to which preeclampsia occurs, or reversing an existing preeclampsia (i.e., reducing the burden of existing misfolded protein aggregates by blocking, degrading, or disrupting misfolded protein aggregates that occur with preeclampsia). These agents, as well as those that have not been evaluated for their effect in treating abnormal protein aggregate-type diseases, can be tested to determine their effect in treating preeclampsia.
Also described herein are methods of treating pregnant women with preeclampsia. One method of treating preeclampsia is to administer to a pregnant woman a therapeutically effective amount of a drug, e.g., one that inhibits (partially or completely) the reformation of protein aggregates; may reverse (partially or fully) the stress of already existing protein aggregates or drug present; drugs that stabilize the original conformation of these proteins and thereby reduce the rate of aberrant folding and subsequent aggregation; or a combination of drugs (more than one drug, the same type of behavior, e.g., two drugs that inhibit protein aggregate reformation; more than one drug, not the same type of behavior, e.g., one drug that inhibits protein aggregate reformation and one drug that reduces the already existing protein aggregate pressure).
1. A method of diagnosing and aiding in the diagnosis of whether a pregnant female has pre-eclampsia comprising:
(a) obtaining a sample from a female; and is
(b) Detecting the presence of a supramolecular aggregate of misfolded protein produced (pathologically, a causative factor) in the sample with a higher probability of the supramolecular aggregate being or will be present in the sample than in a woman without such supramolecular aggregate.
2. A method of diagnosing and aiding in the diagnosis of whether a pregnant female has preeclampsia comprising:
(a) obtaining a sample from a female;
(b) mixing the sample with a reagent capable of labeling supramolecular aggregates of misfolded proteins that (i) contain serpina-1(alpha-1 antitrypsin) or a fragment of serpina-1 and (ii) appear (pathologically, are a factor in the pathogenesis) associated with preeclampsia, under conditions suitable for labeling supramolecular aggregates to occur; and is
(c) Determining whether the sample contains supramolecular aggregates, and if present, the female having or will have a higher probability of pre-eclampsia than if the supramolecular aggregates were not present in the sample.
3.1 the method wherein the supramolecular aggregate comprises serpina-1(alpha-1 antitrypsin) or a fragment of serpina-1.
4.2 or 3 wherein the supramolecular aggregate further comprises at least one of the following proteins: ceruloplasmin, heavy chain of IgG, light chain of IgG and interferon inducible protein 6-16(IFI 6).
5.1, 3 or 4, the presence of supramolecular aggregates is detected by mixing the sample with a reagent that detectably labels the supramolecular aggregates.
6.1-5 is a urine sample or a placental tissue sample.
7.1-6 wherein the reagent is a dye, an antibody or a fluorescent label.
The dye of the process of 8.7 is a heteroaromatic ring dye.
9.8 the dye in the method is Congo red or a thioflavin.
10.7-9 wherein the supramolecular aggregate is stained with a dye and staining of the supramolecular aggregate is detected by dot blot correction and/or spectral shift analysis.
11. In a diagnostic test of whether a pregnant woman has preeclampsia, it is determined whether a woman has congophilic proteinuria and whether a congophilic protein aggregate comprising serpina-1(alpha-1 antitrypsin) or a fragment of serpina-1 can be detected in a urine sample obtained from the pregnant woman.
12.7 wherein the antibody is conformation dependent and amino acid sequence independent.
13.12 the conformation-dependent antibody of the method recognizes one or more epitopes on a supramolecular aggregate of one or more misfolded proteins selected from the group consisting of: pre-fiber oligomers, fibrils, and amyloid fibers.
14.13 the conformation-dependent antibody in the method recognizes one or more epitopes on fiber oligomers and/or amyloid fibers.
15.13 the conformation-dependent antibody in the method binds to one or more epitopes on one fibril (circular).
The conformation-dependent antibody of the method of 16.13 binds to one or more epitopes on a pre-fibrillar oligomer.
17.14 the conformation specific antibody of the method is an OC antibody.
The conformation specific antibody in the method of 18.15 is an Officer antibody.
The conformation specific antibody in the method of 19.16 is the a11 antibody.
20.15 or 18 if the fibril-specific antibody is immunoreactive with the sample, then the woman has suffered from or is about to suffer from a cytolysis.
21. A method of diagnosing or aiding in the diagnosis of whether a pregnant woman has preeclampsia comprising:
(a) Obtaining a urine sample or a placental tissue sample from a pregnant woman;
(b) contacting the sample with a conformation-dependent antibody that binds to supramolecular aggregates of misfolded proteins that occur with preeclampsia, wherein if the antibody binds to a component in the sample, the female is more likely to have preeclampsia or is more likely to have preeclampsia than if the antibody does not bind to the component in the sample.
22.21 the composition of the sample in the method is one of the following polymer aggregates: a pre-fiber oligomer, a fibril, or an amyloid protein fiber.
23.22 of the method of recognizing one or more epitopes on fiber oligomers and/or amyloid fibers.
The conformation-dependent antibody in the method of 24.22 recognizes one or more epitopes on the fibril (circular).
The conformation-dependent antibody in the method of 25.22 recognizes one or more epitopes on the pre-fiber oligomer.
26.23 the conformation specific antibody in the method is an OC antibody.
The conformation specific antibody in the method of 27.24 is an Officer antibody.
The conformation specific antibody in the method of 28.25 is the a11 antibody.
29.24 or 27, if the fibril-specific antibody is immunoreactive with the sample, then the woman has suffered from or is about to suffer from a cytolysis.
30. A method of determining whether a pregnant woman is at risk of preeclampsia comprising:
(a) obtaining a sample from a female;
(b) detecting the presence of supramolecular aggregates of misfolded proteins associated with (pathologically contributing to) preeclampsia in the sample, wherein if supramolecular aggregates are present in the sample, the woman is at greater risk of preeclampsia than if no protein aggregates were present in the sample.
31. A method of determining whether a pregnant woman is at risk of preeclampsia comprising:
(a) obtaining a sample from a female;
(b) mixing the sample with a reagent capable of labeling a supramolecular aggregate of misfolded protein (i) containing serpina-1(alpha-1 antitrypsin) or a fragment of serpina-1 and (ii) associated with (pathologically contributing to) preeclampsia under conditions suitable for labeling supramolecular aggregates; and is
(c) Determining whether the sample contains supramolecular aggregates, and if present, the woman will be at a higher risk of preeclampsia than if no protein aggregates were present in the sample.
32.30 wherein the supramolecular aggregate comprises serpina-1(alpha-1 antitrypsin) or a fragment of serpina-1.
33.31 or 32 wherein the supramolecular aggregate further comprises at least one of: ceruloplasmin, heavy chain of IgG, light chain of IgG and interferon inducible protein 6-16(IFI 6).
34.30, 32 or 33 is detected by mixing the sample with a reagent that detectably labels the supramolecular aggregate.
35.30-34 is a urine sample or a placental tissue sample.
36.34 or 35 is a dye, an antibody or a fluorescent label.
37.36 the dye of the method is Congo red or a thioflavin.
38.35-37 wherein the supramolecular aggregate is stained with congo red and staining of the supramolecular aggregate is detected by dot blot fixation and/or spectral shift analysis.
39. A method of reducing the degree of preeclampsia in a pregnant woman, comprising administering to the woman a therapeutically effective amount of an inhibitor of [ formation ] of protein aggregates in the woman, which aggregates comprise serpina-1(alpha-1 antitrypsin) or a fragment of serpina-1 and are associated with (are a causative factor of) preeclampsia, wherein the therapeutically effective amount is an amount sufficient to inhibit (partially or fully) the formation of supramolecular aggregates, break up (partially or fully) existing supramolecular aggregates, or both.
40.39, the inhibitor stabilizes the native conformation of at least one component of the supramolecular aggregate, thereby reducing the extent to which abnormal folding occurs, such that formation of supramolecular aggregates in a female is reduced compared to the extent in the absence of the inhibitor.
41.39 or 40 is an antibody or antibody fragment that binds a component of a protein aggregate, a trimethylamine N-oxide, an anti-beta amyloid agent (i.e., p-aminophenol, 2-amino-4-chlorophenol and derivatives thereof), a small molecule inhibitor of fibril formation, fibril formation or oligomer formation; or a polypeptide inhibitor of fibril formation, fibril formation or oligomer formation.
42. An in vitro method for diagnosing pre-eclampsia or predicting the future development of pre-eclampsia in a subject comprising detecting abnormal protein aggregates (supramolecular aggregates of misfolded proteins) in a sample of urine or placenta taken from said subject.
43.1 abnormal protein aggregates are detected in a urine sample.
44.42 the abnormal protein aggregates are detected in a placental sample.
45.42-44 the abnormal protein aggregates are detected by Congo red staining.
46.45 the Congo red staining in the method includes dot blot fixation and spectral shift analysis.
47.42-44 the abnormal protein aggregates are detected by thioflavin S or thioflavin T staining.
48. The method of any one of claims 42-47 wherein the abnormal protein aggregate comprises serpina-1(alpha-1 antitrypsin) and/or polypeptide fragments thereof.
49.42-48 the abnormal protein aggregates of any one of the methods include ceruloplasmin fragment and heavy and light chains of IgG.
50. A protein aggregation inhibitor is used for treating and/or preventing preeclampsia.
51.50 inhibits the reformation of protein aggregates.
52.50 or 51 can reverse or break down the formation of existing protein aggregates.
53.50 can stabilize the original conformation of the protein, thereby reducing the rate of aberrant folding and subsequent polymerization.
54.50-53 the inhibitor of any one of claims 50-53 which inhibits the polymerization of serpina-1(alpha-1 antitrypsin) and/or polypeptide fragments thereof is selected from the group consisting of: (a) trimethylamine N-oxide; (b) trimethylamine N-oxide related complex; (c) a FLEAIG polypeptide; and (d) polypeptides related to the FLEAIG polypeptide and derivatives thereof.
55.50-54, is selected from the group consisting of: (a) trimethylamine N-oxide; (b) trimethylamine N-oxide related complex; (c) a FLEAIG polypeptide; and (d) polypeptides related to the FLEAIG polypeptide and derivatives thereof.
56.50-54 is an anti-beta amyloid agent.
57.56 the anti-beta amyloid protein is selected from the group consisting of p-aminophenol and 2-amino-4-chlorophenol.
58.7 the antibody in the method of at least one of: a11 antibody, OC antibody, Officer antibody, M118 antibody, M204 antibody, M205 antibody, M89 antibody (i.e., 89-17), and M09 antibody.
Brief Description of Drawings
FIG. 1 shows a photograph of a nitrocellulose membrane before (left) and after (right) washing after a Congo Red dot test on a urine sample (5 μ L/dot on nitrocellulose membrane).
FIG. 2 shows a photograph of a set of laterally compared urine samples (33 μ L/spot on nitrocellulose) before and after washing after the Congo Red Spot test. The boxed samples are from women with significantly severe preeclampsia.
FIG. 3 is a photograph of a set of longitudinally compared urine samples (33 μ L/spot on nitrocellulose) before and after washing after the Congo Red Spot test. Six pregnant women are tracked in the whole course of the whole pregnancy process, and urine is extracted for a plurality of times for analysis. The samples enclosed by the boxes were from the clinical presentation onset period. The boxes corresponding to samples U348i and U348j are samples taken after a medically indicated delivery (emergency caesarean section) was made due to preeclampsia.
Fig. 4 shows a histogram of Congo Red Retention (CRR) normalized to the total amount of protein in women with severe preeclampsia (sPE), chronic hypertension (cHTN), and normal pregnant Control (CRL).
Fig. 5 shows ROC curves for Congo Red Retention (CRR) and Congo Red Integration (CRI) respectively with (a) a diagnosis of preeclampsia and (B) a co-factor predictive of forced delivery due to preeclampsia (233 urine samples from 114 different pregnant women).
Figure 6 shows a graph correlating Congo Red Retention (CRR) of urine protein and the presence and severity of preeclampsia as determined from abnormal proteomic data (UPSr).
Fig. 7 shows the Congo Red Retention (CRR) and the ratio index uFP of urine protein: graph of log [ sFlt-1/PIGF × 100] correlation.
FIG. 8 shows a method for separating misfolded urinary proteins in the pre-eclampsia stage by (A) dot blot detected Congo Red affinity, (B) gel filtration or (C) centrifugation. The framed samples were from women with clinically overt pre-severe eclampsia (sPE).
FIG. 9 shows a graph illustrating the% Congo Red Retention (CRR) of urinary protein for different disease state groups (CRL: control; cHTN: chronic hypertension; gHTN: gestational hypertension; mPE: Mild preeclampsia; sPE: Severe preeclampsia; spPE: overlapping preeclampsia) enrolled in (A); (B) (ii)% Congo Red Retention (CRR) of urine protein for different delivery groups (IND: indicated delivery); (C) an accuracy map comparing (1)% Congo Red Retention (CRR) for urine protein, (2) the ratio of sFLT/PIGF in urine, and (3) the prediction accuracy (sensitivity/100-specificity) of the P/C ratio, i.e., urine protein/creatine ratio.
FIG. 10 shows a photograph of a Western blot experiment performed on nitrocellulose membrane using spots of urine samples (shown: urine, blood, cerebrospinal fluid (CSF) and placental lysate; PE: preeclampsia; CRL: control), probed with three polyclonal antibodies A11, OC and Officer.
FIG. 11 shows a photograph of a Western blotting experiment on a nitrocellulose membrane using a spot of a urine sample (PE: preeclampsia; CRL: control; cHTN: chronic hypertension) detected with polyclonal antibody A11. Red arrows mark the position of the sample spots in different rows on the film. The film was compared to the sample lattice array and each sample position circled by a black circle.
FIG. 12 shows a photograph of a Western blot experiment performed on nitrocellulose membrane with a spot of a urine sample (PE: preeclampsia; CRL: control), probed with polyclonal antibody A11(A11, column 1), and the primary A11 antibody was discarded in the negative control (neg., column 2) to see if there was non-specific binding of the secondary antibody.
FIG. 13 shows a photograph of a Western blot experiment performed on nitrocellulose membrane with a spot of a urine sample (PE: preeclampsia; CRL: control), probed with monoclonal antibodies (M204, M205, M118, M89-17, M09, M55) and polyclonal antibody A11(A11), and the primary A11 antibody was discarded in the negative control (neg., column 2) to see if there was non-specific binding of the secondary antibody.
Figure 14 sets forth a graph showing the prediction of an indicated labor resulting from preeclampsia in a longitudinally tracked group of patients.
Figure 15A shows placental slices from three women, two of which were preterm because of severe preeclampsia (a-F) and the other was primary preterm (control, G-H), both sections stained with congo red and viewed under a microscope with white light (a, D, G) or polarized light (B, C, E, F, H and I). Panels C and F are enlargements (640x) of the box areas in panels B & E. FIG. I: polarized light images of brain sections from a patient with senile dementia stained with congo red in the same conditions.
FIG. 15B shows an image of Congo Red Positive sediment in urine of a patient before eclampsia.
FIG. 16 shows a double reducing SDS PAGE electrophoresis of urine samples from two women with severe preeclampsia. The left panel shows the gel after staining all proteins with Coomassie Brilliant blue (columns 1-4). The right panel shows the immunoreactivity of serpin a1 transferred to protein on nitrocellulose membrane. Molecular weight tags (MW) are presented in each figure.
Figure 17 shows the results of a secondary screen for anti-APF monoclonal antibodies 09 and 89.
FIG. 18 shows the results of the monoclonal antibody 09 and 89 analysis against alpha hemolysin and ABeta.
Detailed Description
Preeclampsia occurs during the latter half of pregnancy and is associated with morbidity or mortality in a large number of pregnant women and fetuses. There is currently no effective screening method to diagnose or assess the risk of developing this disease and the associated hypertension. As a result, pregnant women are not effectively monitored or treated until the disease causes other complications, including elevated blood pressure, urine appearing proteins, and the like. In addition, women who are not at risk for such diseases are also unnecessarily checked for symptoms throughout pregnancy because there is no effective way to distinguish them from the at-risk population early in pregnancy. Moreover, current testing methods require supervision by a physician.
The methods and compositions described herein for detecting and/or monitoring congophilic properties associated with preeclampsia and the specific configuration of misfolded protein aggregates that are associated with or cause preeclampsia are useful in the diagnosis and treatment of preeclampsia. Also described herein are additional biomarkers that can be used independently to diagnose and treat preeclampsia, and in association with the methods and articles described herein, these markers can also be used to further confirm and assess the status of preeclampsia in pregnant women. For example, by detecting misfolded protein aggregates, a woman who is judged by the above method to have preeclampsia or may be at high risk of developing preeclampsia may be further evaluated by methods based on additional biomarkers or indicators that are correlated (indicative) for preeclampsia. This includes, for example, detecting the presence or absence of a particular preeclampsia-related (indicative) biomarker in the urine of a pregnant woman (e.g., Serpins a1(serpin a1) and albumin), and/or measuring the ratio of a particular preeclampsia-related (indicative) biomarker in urine (e.g., sFlt and PIGF). The assessment of these biomarkers can confirm or assist in confirming (inverting or assisting in inverting) the results of the above method, i.e., whether a pregnant female has or is at risk of developing preeclampsia. Can be prepared byMethods for performing such evaluations are described herein and reference is made to the following patent disclosures (e.g., U.S. patent application No. 12/084004; PCT/US2006/042585 and U.S. patent application publication No.: US-2006-0183175; PCT/US2005/047010, the entire contents of which are incorporated herein by reference).
The above-mentioned method may be used for diagnosing or aiding in the diagnosis of whether a pregnant woman suffers from, or is at high risk of suffering from, the following hypertensive disorders: preeclampsia, eclampsia, mild preeclampsia, chronic hypertension, EPH gestational toxicosis, gestational hypertension, superimposed preeclampsia (including preeclampsia superimposed with chronic hypertension, chronic kidney disease, or lupus), HELLP syndrome (hemolysis, elevated liver enzymes, decreased platelet content), and nephropathy.
The methods described herein may also be used to assess the risk of a pregnant woman suffering from a particular hypertensive complication, including preeclampsia. These complications may include caesarean section, elevated plasma uric acid, elevated systolic and diastolic blood pressure, protein response in the dipstick, number of pregnancies, abnormal fetal weight at birth, placental rupture, IUGR, hemolysis, thrombocytopenia, elevated liver enzymes and HELLP syndrome (hemolysis, elevated liver enzymes, reduced platelet content).
1. Congo red test/congo red preference for diagnosis of preeclampsia
The destabilization, misfolding and aggregation of proteins are supramolecular structures with affinity (congo red-avid) for the self-assembling dye congo red, a common feature of an increasing number of a series of progressively-developing human diseases, such as alzheimer's disease, parkinson's disease and raney virus disease. The affinity for the azo dye Congo Red (CR) was used to detect abnormal amyloid aggregation in diseases caused by misfolding of this protein. The applicant has made new and unexpected findings: preeclampsia is a specific disease of pregnant women associated with protein misfolding and protein aggregation. They demonstrated that preeclampsia is characterized by supramolecular amyloid aggregation and congo red urine protein, indicating increased secretion of misfolded proteins. The applicant speculates that the congo red-philic associated with preeclampsia in urine provides a diagnostic and/or prognostic diagnostic test for preeclampsia by measuring the degree of misfolding of the mesoglobulin.
Misfolded proteins associated with preeclampsia can be found in "supramolecular misfolded protein aggregates". Herein, the term "supramolecular misfolded protein aggregates" (also referred to herein as "supramolecular aggregates", "abnormal protein aggregates", "supramolecular amyloidogenic protein aggregates" and "congo red protein aggregates") refers to both soluble protein aggregates and insoluble protein aggregates. These include: (1) fibrillar precursor oligomers (both "amyloid precursor oligomers" and "non-fibrillar misfolded protein aggregates" only), soluble and with "amyloid" properties; (2) fibrils; (3) a fiber oligomer, soluble; and (4) amyloidogenic fibers, insoluble.
Samples, e.g., urine samples, obtained from preeclamptic patients, patients who may have preeclampsia, and pregnant women at risk for preeclampsia may contain relatively little or no insoluble amyloidogenic fibers, but may contain a mixture of one or more soluble supramolecular protein aggregates, e.g., fibrillar precursor oligomers, fibrillar oligomers, and/or fibrils.
In some embodiments, a method for diagnosing preeclampsia includes detecting a supramolecular protein aggregate in a sample. Supramolecular aggregates can be detected using, for example, heteroaromatic rings and/or fiber-specific dyes. In some embodiments, supramolecular aggregates in a sample (e.g., in a urine sample) are detected by using properties of the supramolecular aggregates that increase/enhance fluorescence (or cause spectral shifts) of heteroaromatic dyes, including, for example, thioflavins (thioflavins T, ThT or thioflavins S, ThS) and Congo Red (CR), as compared to common proteins. In some embodiments, the method of diagnosing preeclampsia comprises detecting soluble supramolecular aggregates, e.g., detecting fiber precursor oligomers in a sample using heteroaromatic rings and/or fiber-specific dyes. We do not wish to be bound by a particular theory, but we believe that the addition of heteroaromatic rings and/or fibre-specific dyes to soluble supramolecular aggregates may promote the formation and precipitation of insoluble supramolecular aggregates, such as fibre precursor aggregates and/or fibre aggregates, so that these aggregates become readily detectable.
Provided herein are methods of diagnosing or aiding in the diagnosis of pre-eclampsia. In some embodiments, detection of urine congophilic (e.g., using dot blot fixation or spectral shift analysis) is indicative of whether a pregnant woman has preeclampsia. In some embodiments, the above method is used to detect congophilic protein aggregates in urine, not only as diagnostic evidence of pre-eclampsia that has already developed, but also to predict whether a pregnant woman is likely to develop pre-eclampsia in the future. In some embodiments, congo red-tropic protein aggregates stained by congo red in placental tissue can be used to diagnose pre-eclampsia. It will be appreciated that protein aggregates can be detected by a variety of other methods and reagents. These are within the ordinary skill and require undue experimentation and are part of and are included herein.
In addition to dyes such as congo red and thioflavin, other reagents (including congo red and thioflavin derivatives) may also be used to detect the supramolecular protein aggregates described herein. Such detection reagents include, but are not limited to: curcumin analogues (J.Am.chem Soc.131:15257 (2009); X-34(1,4-bis (3-methoxy-4-hydroxyphenylethenyl) -bezene), a highly fluorescent congo red (Ikonomovic et al, Methods in Enzymology 412(2006), "Amyloid, Prions and other protein aggregates", Part B pp:123-144) and J.Histochem and Cytochem 48:1223 (2000); Thioflavin S, Thioflavin T; Nile Red; Acridine orange; amino-8-naphthalenesulfonate (ANS) and Bis-ANS; 4-dicyanoethyleneLonelin (DCVJ) (Biophysic Journal 94(12):4867-4879, 2008); AO1987 (oxazine dyes) (Nature Biotechnology 23(5)577 (2005)); fluorescent styrene dyes (Angewandte Chemie, Internnatio)nal Edition 43(46) 6331-; BF-168: (6-2-Fluoroethoxy) -2- [2- (4-methylaminophenol) ethenyl]Benzoxazole (j. neuroscience 24(10),2535 (2004)); BSB: (trans ) -1-bromo-2, 5-bis- (3-hydroxycarbonyl-4-hydroxy) styrylbenzene (lab. investigation 83(12)1751 (2003)); quinolinhydrazone compounds (e.g., 4-methyl-7-methoxy-2- (4-quinolinylmethylenehydrazine) quinoline) (WO2002/024652, Thomas Raub et al, Chrisimin G et al Chrysamine-G, a lipophilic analog of Congo red, inhibitors A beta-induced toxicity in PC12cells. Life Sci.1998; 63(20): 1807-14); rifampicin (Tomiyama T et al. J Biol chem. 1996; 271: 6839-6844); melatonin (Pappolla Met al.J Biol chem.1998; 273: 7185-7188); baicalein (Zhu M et al, the flavoid basic inhibition hybridization of alpha-synuclein and disperggregates experimental hybridization. J Biol chem.2004Jun 25; 279(26): 26846-57); scyllo-inositol (scylla inositol, 1,3,5/2,4,6-Hexahydroxycyclohexane (Cocositol, Quercinitol,1,3,5/2,4,6-Hexahydroxycyclohexane)) and its derivatives (Sun et al. Synthesis of scyllo-inositols and the effects on alkyl oil beta peptide aggregation. bioorging Med. chem.2008Aug 1; 16(15): 7177-84); imaging probes, e.g. [11C ] ]-PIB: n-methyl [11C]2- (4' -methylaminophenol-6-hydroxybenzotriazole) (Brain 130:2607 (2007)); stilbene-based benzotriazoles and derivatives thereof (Bioorganic and Medicinal Chemistry Letters 18:1534(2008)) and other reagents such as the examples described in FEBS Letters 583:2593 (2009).
In some embodiments, a non-invasive and rapid urine diagnostic method is provided. In some embodiments, these diagnostic methods are based on congo red affinity. In some embodiments, the total degree of protein misfolding in a preeclamptic patient may be determined by a simple diagnostic test (urine Congo Red Retention (CRR)). The CRR test may also be used to predict IND, an important cause of preterm birth.
The methods and compositions described herein allow one to assess and/or monitor the risk of a pregnant woman suffering from hypertension, such as congo red retention, by detecting and/or monitoring congo red preference. It is to be understood that other dyes that can react/bind to protein aggregates, and/or dyes with congo red-like chemistry, can also be used in the above method. The present invention is not limited to the use of congo red.
In some embodiments, the diagnostic method comprises obtaining a protein sample from a pregnant female and testing the protein sample for congo red-philic. In some embodiments, congo red-philic is detected by contacting the urine sample with congo red dye for a sufficient time to allow congo red to bind (CRI), followed by washing the sample with a wash solution. After washing, the sample Congo Red Retention value (CRR) indicates whether the pregnant woman has or is at risk of developing preeclampsia. As a reference for Congo Red binding values, positive and negative controls can be introduced, e.g., samples obtained from patients who have been diagnosed with preeclampsia, and samples obtained from normotensive populations. The binding step and/or the washing step may be compared by adding positive and negative controls. A positive sample that still retains congo red staining after washing (CRR positive) indicates adequate staining, and a negative sample that does not contain congo red after washing indicates adequate washing (CRR negative). In some embodiments, the CRR can be visualized without visual instrumentation, e.g., by separation methods such as congo red affinity dot blotting, gel filtration, and centrifugation, as well as washing of precipitated congo red binding proteins (see fig. 8). In some embodiments, the separation step may be performed in a container or tube, such as Falcon TM Or Eppendorf TM The tube, dye binding and washing may be performed in this container or tube, followed by centrifugation to separate (e.g., precipitate) protein-bound dye from unbound protein dye, which may then be selected as an additional washing step. In some embodiments, the sample can be spotted on a membrane with protein affinity, such as a nitrocellulose membrane, and the staining and washing steps can be performed on the membrane (e.g., dot blotting). Such tests may require limited or no supervision by a trained physician, e.g., performed by a trained technician, or in some cases by the patient, without the need for guidance and/or laboratory equipment (e.g., using a kit containing the necessary reagents and instructions).
Applicants have found that despite the fact that the protein concentration in urine samples from a group of pregnant women is far apart, the protein levels in urine are generally sufficient to measure congo red preference without the need to concentrate the sample. This allows one to design simple analytical tests that require no special requirements for laboratory instrumentation, or only basic instrumentation.
In some embodiments, the percentage congo red binding value (% CRI) can be determined, for example, by using optical density measurements (OD, densitometer) compared to a negative control, and multiplied by 100. The percentage congo red retention after washing (% CRR) can also be measured using Optical Density (OD), and the results compared to the negative control and multiplied by 100. The CRR value of the sample can be obtained by comparison with the corresponding negative control value. An adequate wash is a sign that there is no visible residual congo red in the negative control and the mean OD should be 0 (if multiple controls are used).
In some embodiments, Congo Red Retention (CRR) and congo red binding (CRI) coefficients can be calculated, samples with CRR > 20% can be "referred to as" red spot test positive, while those with CRR < 15% are negative.
In some embodiments, detection of congophilic urine protein in urine using the diagnostic methods described herein can detect that a pregnant woman has severe pre-eclampsia between 8 and 10 weeks prior to the appearance of clinical characterization. In some embodiments, identification of urocongophilic (by dot blot fixation and/or spectral shift assays) is indicative of whether a patient has preeclampsia and a method for diagnosing preeclampsia is provided. In some embodiments, the urinary congo red protein aggregates detected by the above methods can be used not only to diagnose existing preeclampsia, but also to predict the likelihood of future development of preeclampsia.
In some embodiments, the urine sample may be analyzed immediately after collection (e.g., on a dot blot, urine test stick, or other similar item), or may wait for a period of time. For example, the urine sample may be frozen at-70 degrees Celsius and/or collected in a tube or container and dried and stored at-20 degrees Celsius or 4 degrees Celsius. Drying of the sample may use vacuum centrifugation (e.g., SpeedVac), and the dried sample may be stored longer and reduce the risk of deterioration (e.g., protein denaturation) when analyzed above 0 degrees celsius. It should be noted that it is generally not necessary to concentrate the sample by drying, but the sample may be dried for the reasons described above or otherwise.
2. Diagnosis of preeclampsia using conformation-specific protein aggregate antibodies
Applicants have unexpectedly discovered that preeclampsia is a protein conformation disease associated with protein misfolding and aggregation, and that one of its important features is amyloid aggregation.
We here detail several methods for diagnosing preeclampsia, which use specific antibodies to identify certain pre-eclampsia-characteristic protein aggregates in the urine of pregnant women, and thereby diagnose the disease.
The methods described herein use polyclonal or monoclonal antibodies to detect protein conformations associated with protein aggregation-type disorders such as preeclampsia, among which are: (i) detecting a pre-fibrillar soluble oligomer conformation, such as the "A11" antibody used in this patent; (ii) detecting a cyclic fibril conformation, such as the "Officer" antibody used in this patent; (iii) a fine fiber-like conformation is detected, for example, as used in this patent, an "OC" antibody, etc.
In the present description, "misfolded protein supramolecular aggregates" (also referred to as "supramolecular aggregates", "abnormal protein aggregates", "supramolecular amyloid aggregates" or "congo red protein-philic aggregates") refer to both soluble and insoluble protein aggregates. These aggregates include: (1) pre-fibrillar oligomers (also known as "pre-amyloid oligomers" or "non-fibrillar misprotein aggregates") that are soluble and have the characteristics of "amyloid"; (2) fibrillar aggregates; (3) fibrous oligomers, such aggregates also being soluble; (4) amyloid fibrils, such aggregates are insoluble.
Each of these supramolecular aggregates is capable of being recognized by specific antibodies, such as the conformation-dependent-sequence-independent antibodies mentioned in the present description.
Protein aggregates having "amyloid" characteristics referred to herein have certain common chemical, physical, biological characteristics, all of which comprise amyloid fibrils, but may differ in other chemical, physical, biological characteristics, e.g., for certain amyloid protein aggregates, their structure may differ.
In certain embodiments, the pre-fibrillar oligomers are present in the urine of a patient suffering from preeclampsia. In certain embodiments, the presence of such pre-fibrillar oligomers in a sample, e.g., in urine, can be recognized by a conformation-dependent-amino acid sequence-independent antibody. In certain embodiments, the a11 antibody may play this role.
In certain embodiments, the fibrils will be present in a sample, such as urine from a preeclamptic patient. In some cases, the presence of these fibrils in a sample, such as urine, can be recognized by conformation-dependent-amino acid sequence-independent antibodies. In certain embodiments, the Officer antibody may serve this role.
In certain embodiments, fibrillar oligomers or amyloid fibrils are present in a sample, such as urine from a preeclamptic patient. In some cases, the presence of these fibrillar oligomers or amyloid fibrils in a sample, such as urine, can be recognized by a conformation-dependent-amino acid sequence-independent antibody. In certain instances, OC antibodies can play this role.
Such an antibody recognizing supramolecular aggregates conformation-dependent amino acid sequence-independent can be prepared using animals, e.g. mice or rabbits, with polypeptide fibrils or fibers, fibrillar aggregates, fibrils or amyloid fibers, etc. containing pro-fibrillar aggregates (oligomers), having "amyloid" properties and possibly forming such pro-fibrillar aggregates. Some polypeptides having "amyloid" characteristics are now known, such as A.beta.1-40, A.beta.1-42) Polyglutamine (PolyGln) molecule NH2-KKQ 42 KK-COOH, etc., as exemplified in Example 4, Kaied et al. mol neurogene.2007; 2: 18; hrncic et al.am J Pathol.2000; 157: 1239-1246; o' Nualliin B et al Proc Natl Acad Sci USA.2002; 99: 1485-.
The animal from which the antibody is prepared may be immunized with a population of morphologically pure fibers, for example, to produce a conformation-dependent amino acid sequence-independent antibody that specifically recognizes fibers or fibrillar aggregates. Some of the antibodies so produced may specifically recognize fibers or fibrillar aggregates, but not random helical monomers, pre-fibrillar oligomers, or normally folded precursor proteins (not cross-reactive).
Another approach is to immunize animals with amorphous pre-fibrillar aggregates to produce antibodies, e.g., to produce conformation-dependent-amino acid sequence-independent antibodies that specifically recognize pre-fibrillar aggregates. Some of the antibodies so produced may specifically recognize pre-fibrillar aggregates and not (cross-react) fibrillar oligomers, amyloid fibrils, monomers or normally folded precursor proteins.
Another approach is to immunize animals with fibrils to produce antibodies, for example to produce conformation-dependent-amino acid sequence-independent antibodies that specifically recognize circular fibrils. Some of the antibodies so produced may specifically recognize fibrils, but not (without cross-reactivity) pre-fibrillar oligomers, amyloid fibrils, monomers, or normally folded precursor proteins.
Applicants have demonstrated that the specificity of detecting abnormal protein conformations (supramolecular aggregates) associated with preeclampsia can increase significantly as secondary antibodies are pre-adsorbed by human IgG to reduce non-specific binding. Part of this conclusion is based on the applicants' discovery that supramolecular aggregates of proteins associated with preeclampsia comprise human immunoglobulins or fragments thereof. The crosslinking reaction of the secondary antibody that is not adsorbed in advance may result in non-specific binding of the secondary antibody. In certain embodiments, methods are provided for detecting a pre-eclampsia-related abnormal protein conformation in urine, comprising obtaining a urine sample from a pregnant female who has suffered from, is suspected of suffering from, or is at high risk of developing pre-eclampsia, contacting the urine sample with a primary antibody (monoclonal or polyclonal) that specifically recognizes a conformation associated with a protein aggregation-type disease under conditions suitable for binding, allowing binding of misfolded protein to the antibody to form a composition, contacting the combination with a secondary antibody that has been previously adsorbed with human IgG to reduce non-specific binding, and detecting the presence of a complex of misfolded protein and antibody in the urine, the presence of such a complex indicating that pre-eclampsia is likely to occur.
In some embodiments, the protein aggregate is contacted with a11 primary antibody, and then with a pre-adsorbed secondary antibody, according to the methods above. Thus, the degree of positivity of A11 detected is indicative of the severity of preeclampsia syndrome.
It should be understood that any secondary antibody capable of detecting the primary antibody may be used as long as it is capable of being pre-adsorbed to human IgG sufficiently to prevent non-specific binding. In this respect, the invention is not limited to secondary antibodies. The secondary antibody may be linked to any label for detection, including but not limited to a radioisotope label, an enzymatic label, a non-radioisotope label, a fluorescent label, a toxin label, an affinity label, a chemiluminescent label, or a nuclear magnetic resonance contrast agent, among others. It should also be noted that the detection system can be easily varied according to the purpose, such as high throughput/automated screening using various labels or other detection methods, and the invention is not limited in this respect. It should also be understood that the antibodies mentioned herein are by way of example only and that further antibodies may be generated by the methods mentioned herein or by other known methods. Other antibodies than those mentioned herein, including but not limited to whole antibodies, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, human antibodies, primate antibodies, multispecific antibodies, single chain antibodies, epitope binding antibodies, antibodies containing a VL or VH domain, and the like, e.g., antibodies that recognize protein aggregates, recognize misfolded or abnormal protein conformations, recognize fragments of specific proteins (those biomarker proteins mentioned in this patent), and the like, also form part of this invention.
In some embodiments, the invention provides monoclonal antibodies that recognize protein aggregates, misfolding or abnormal protein conformation. In certain embodiments, antibodies prepared using the same antigen as the A11 antibody are provided (Science300:486-489,2003) that preferentially recognize different classes of pre-fibrillar oligomer conformations. In certain embodiments, monoclonal antibodies #204, #205, and #89 are provided that are shown to immunologically recognize protein aggregates, recognize such protein aggregates in urine from a preeclamptic patient, and detect the absence of such protein aggregates in urine from a control group. In certain embodiments, monoclonal antibodies that recognize different conformations of the pre-fibrin oligomers, such as the #204, #205, and #89 monoclonal antibodies, may be used to diagnose pre-eclampsia.
The protein aggregates in urine referred to herein may not only be used to diagnose pre-eclampsia that has already occurred, but may also be used to predict the future occurrence of this disease. In addition to detecting the presence of protein aggregates in urine, placental tissue samples may also be characterized by the presence of such protein aggregates. Methods for detecting abnormally folded proteins and protein aggregates in the placenta using selective antibodies can also be used to diagnose preeclampsia. In certain embodiments, antibodies that bind to protein oligomers may have therapeutic effects, such as (i) disruption of protein oligomers/aggregates; (ii) preventing further growth of protein oligomers/aggregates; (iii) stabilize existing protein oligomers/aggregates from converting to more pathogenic conformations; (iv) the protein oligomers/aggregates are converted from a pathogenic conformation to a non-pathogenic conformation.
Such therapeutic methods have been used for the treatment of senile dementia, for example, using an anti-beta-amyloid antibody and the like. Applicants have discovered that preeclampsia is a disease associated with the accumulation of abnormal protein oligomers. In some embodiments, antibodies having affinity for the unique conformation of the aberrantly folded protein, such as the #204, #205, and #89-17 monoclonal antibodies or similar antibodies mentioned in this patent, can be used to treat preeclampsia.
Applicants have discovered that protein aggregates present in the urine of preeclamptic patients, including one or more of immunoglobulin heavy and light chains, ceruloplasmin and interferon protein 6-16(IFI-6), are also of interest for detecting, preventing and treating this disease. In some embodiments, diagnostic and prognostic methods for preeclampsia are provided that include quantitative analysis of proteins in protein aggregates in the placenta or urine of preeclampsia patients, the corresponding proteins including immunoglobulin heavy and light chains, ceruloplasmin, and IFI-6 protein. In some embodiments, the quantitative analysis for proteins includes measuring protein concentration relative to a standard, such as a sample from a normotensive individual. In some embodiments, antibodies that specifically recognize immunoglobulin heavy and light chains, ceruloplasmin, or IFI-6 are used.
In some embodiments, antibodies that specifically recognize a conformational epitope of misfolded protein aggregates present in a urine sample of a preeclamptic patient or high risk individual are provided for detecting the onset of preeclampsia. Antibodies provided herein include monoclonal and polyclonal antibodies, as well as antibody fragments and antibody derivatives comprising the relevant antigen recognition domain. The term "antibody" refers to an immunoglobulin molecule or other molecule that contains at least one antigen binding domain. The term "antibody" as used herein includes whole antibodies (e.g., IgG, IgA, IgE, IgM or IgD), monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, primate antibodies, multispecific antibodies, single chain antibodies, epitope-binding fragments such as Fab, Fab 'and F (ab') 2, Fd, Fvs, single chain Fvs (scFv), sulfur-crosslinked Fvs (sdFv), fragments containing VL or VH domains, and fully synthetic or recombinant antibodies.
The immunoglobulin or antibody molecules of the invention may be of any class (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecules.
In some embodiments, polyclonal antibodies, such as "Office", "OC" and "a 11" polyclonal antibodies, are provided. Monoclonal and polyclonal antibodies can be obtained from living organisms by immunization with different epitopes of the immunogen, for example with oligomeric protein aggregates (pre-fibrillar and fibrillar), fibrillar aggregates or aggregates containing amyloid fibrils. These aggregates may be uniform in conformation or heterogeneous/amorphous.
The anti-plasma can be obtained from a series of animals by one or more injections of an antigen, and optionally, non-specific immunopotentiators such as adjuvants and the like can be added. For many small molecules or haptens, it may be desirable to link to a carrier protein via a coupling reagent that may be recognized by helper T cells to aid in their function. Homogeneous or heterogeneous/amorphous oligomeric protein aggregates (pre-fibrillar or fibrillar), fibril aggregates or aggregates containing amyloid fibrils may be used in conjunction with an adjuvant, such as incomplete freund's adjuvant.
After one or more immunizations, the antibodies produced may be predominantly IgG with affinity for the epitope. Polyclonal antibodies can provide a variety of specificities. The specificity of polyclonal antibodies can be improved by affinity chromatography with purified antigen.
In some embodiments, monoclonal antibodies, such as "M204," "M205," and "M89" monoclonal antibodies are provided. These and other monoclonal antibodies recognizing similar antigens can be used in the methods described in this patent, such as to diagnose or aid in the diagnosis of preeclampsia; predicting or aiding in predicting the likelihood of a pregnant female developing preeclampsia; and determining or assisting in determining whether a pregnant woman is at risk of developing preeclampsia.
For example, the extent of the immune response of monoclonal antibody 89 to urine from pregnant women can indicate the risk and severity of HELLP syndrome, an atypical form of severe preeclampsia characterized by hemolysis, elevated liver enzyme levels, and low platelet levels. In HELLP syndrome, early diagnosis is very important, as the lethality of this condition is as high as 25%. Monoclonal antibodies against alpha fibrils (alpha protofibrils) have been prepared which are capable of reacting with fibrils formed by hemolysin, these aggregates being formed by exotoxins produced by bacteria and being capable of causing erythrocyte lysis in vitro experiments. The results provided in this patent application (e.g., figure 13) support the use of the relative immunoreactivity intensity of monoclonal antibody 89 to either monoclonal antibody 204, 205 or polyclonal antibody a11 to determine the risk of HELLP syndrome and the extent of acute need for treatment. Also described are methods of determining or assisting in determining the risk of or extent of acute treatment for pregnant women with HELLP syndrome. This method involves evaluating the ratio of the immunoreactive intensity of monoclonal antibody 89 to the reactivity of at least one of monoclonal antibodies 204, 205 and polyclonal antibody a11 in a sample (e.g., urine or placental tissue). This ratio of response intensities, in contrast to control pregnancies not suffering from preeclampsia, can determine or assist in determining the likelihood of a pregnant woman suffering from HELLP syndrome.
Monoclonal antibodies can be obtained by immunizing animals, such as mice and rats. B cells can be isolated from the immunized animal, for example, from the pancreas. The isolated B cells can be fused with other cells, such as bone marrow cancer cell lines, to produce hybridoma cells that can be cultured in an ex vivo environment indefinitely. These hybridoma cells can be isolated by dilution (single cell cloning) and cultured into different colonies. These colonies can be used to screen individual colonies for antibodies that produce a particular affinity and specificity. The selected hybridoma cells can be cultured in a culture medium, and the produced antibody can be isolated from the culture medium. Hybridoma cells can also be injected into an animal, such as a mouse, to produce a live tumor (e.g., a peritoneal tumor) and thereby produce antibodies, which can be collected from ascites fluid. The cell lysis-promoting effect of the plasma can be selectively inactivated, for example by heating.
Examples of antibodies that can be used to make antibodies include certain specific proteins, polypeptides, haptens, chemical compounds and protein aggregates, such as homogeneous and heterogeneous/amorphous oligomeric aggregates (pre-fibrillar and fibrillar), fibrillar aggregates or aggregates containing amyloid fibrils. One knowledgeable of this approach will find that the amount of polypeptide used for immunization will vary depending on a number of factors, such as the animal used for immunization, the antigenicity of the selected polypeptide, and the site of injection. The polypeptides (e.g., aggregates) used as immunogens may be appropriately modified or used in conjunction with adjuvants to increase the antigenicity of the polypeptides. In some cases, polypeptides (aggregates), peptide chains, haptens and small molecules may be conjugated to carrier proteins to elicit an immune response. Homogeneous or heterogeneous/amorphous oligomeric aggregates (fibrils ), fibrillar aggregates or aggregates containing amyloid fibrils may be used in conjunction with an adjuvant, such as incomplete freund's adjuvant.
Methods for increasing antigenicity are known, and include, for example, linking the antigen to a heterologous protein (e.g., globulin or. beta. -galactosidase), or adding an adjuvant during immunization.
The amount of antibody to be used can be determined by titration, for example, by antigen-specific ELISA, Western blot analysis, or radioimmunoassay. Antibodies are typically produced using one or more animals. The antibodies or immunospecific fragments provided herein may be from any of the following animals: including rabbits, sheep, goats, chickens, mice, rats, hamsters, guinea pigs, donkeys, camels, or horses.
Following one or more injections of the antigen, 7-10 days after each injection, animal plasma can be taken to determine the amount of production of a particular antibody (titration). Such a test may be for the antigen itself, for example by ELISA. The antibodies can be stored in a number of different buffered solutions, for example, 0.01M at neutral pH, pH7.4 Phosphate Buffered Saline (PBS), optionally with the addition of 0.1% sodium azide to inhibit microbial growth. For long term storage, the antibody may be stored at low temperatures, e.g.4 ℃, -20 ℃ or-70 ℃. The antibody may be stored at a concentration of greater than 0.5mg/mL, a carrier protein (e.g., 1% bovine plasma albumin) may be added, or 50% glycerol may be added if freezing is desired.
Methods for preparing antibodies, including preparing antigens, immunizing animals, and collecting anti-plasma can be found in the following references: antibodies A Laboratory Manual, E.Harlow and D.Lane, ed., Cold Spring Harbor Laboratory (Cold Spring Harbor, NY,1988) pp.55-120 and A.M.Campbell, Monoclonal Antibody Technology; laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1984).
The term "antibody fragment" as used herein includes any antibody comprising an antigen binding domain that exhibits antigen binding function. Antibodies can be fragmented using conventional techniques. For example, treatment of an antibody with pepsin can produce F (ab') 2 fragments. The F (ab ') 2 fragments produced can be treated to reduce disulfide bonds to produce Fab' fragments. Papain digestion can form Fab fragments. Recombinant techniques or chemical synthesis can be used to produce Fab, Fab ', F (ab') 2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other antibody fragments. Such methods of producing antibody fragments have been described in detail. Antibody fragments, including single chain antibodies, may comprise only the variable regions, and may also contain combinations of the following regions, in whole or in part: hinge region, CH1, CH2 and CH3 domains.
In some embodiments, the antibody or antibody fragment contains one antibody light chain variable region (VL) and one antibody heavy chain variable region (VH) that typically comprises an antigen binding site. In certain instances, the antibody or antibody fragment contains all or part of a heavy chain constant region, such as an IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE, IgM, or IgD constant region. In some cases, the heavy chain constant region is all or part of the heavy chain constant region of IgG 1. The antibody or antibody fragment may also contain all or part of a kappa light chain constant region, or a lambda light chain constant region. In some cases, the light chain constant region is a lambda light chain constant region, or a portion thereof. All or part of these constant regions may be naturally occurring or may be partially or wholly synthetic. The sequence of these constant regions has been reported in detail.
The term "heavy chain portion" as used herein includes amino acid sequences from immunoglobulin heavy chains. A polypeptide comprising a heavy chain portion comprises at least one of: a CH1 domain, a hinge domain (higher, middle or lower hinge domain), a CH2 domain, a CH3 domain, or fragments and derivatives thereof. For example, the polypeptide chain used for conjugation in this patent may contain a CH1 domain; or a CH1 domain, at least part of a hinge domain, and a CH2 domain; or a CH1 domain and a CH3 domain; or a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In other embodiments, the polypeptide chain of this patent contains a CH3 domain. Further, a polypeptide chain as used herein may lack at least a portion of a CH2 domain (e.g., all or part of a CH2 domain). It is understood from the basic knowledge that these domains (e.g., heavy chain portions) may be modified to differ from the amino acid sequence of the native immunoglobulin.
The term "light chain portion" as used herein encompasses amino acid sequences derived from a native immunoglobulin light chain. Typically, the light chain portion contains at least one VL or CL domain.
In some embodiments, the antibody or antigen-binding fragment is a mammalian antibody or antigen-binding fragment, such as a mouse, rat, rabbit, human antibody or antigen-binding fragment.
In some embodiments, the antibody used is a human antibody. "human" as used herein to describe an antibody means that the antibody or fragment contains variable (e.g., VH, VL, CDR or FR regions) or constant regions or both derived from or identical to a human (e.g., derived from a human germ cell or somatic cell).
In some embodiments, the humanized or human antibodies can be used in human therapy, e.g., to treat or prevent preeclampsia. In these antibodies, the effector moiety may be of human origin, so the antibody may interact better with other parts of the human immune system, i.e. the antibody will not be recognised by the body as a foreign substance. In some cases, these antibodies have the same half-life as human antibodies in the presence of solar heat.
In some embodiments, a human antibody may contain one or more sequences encoded by human nucleotides that are not naturally occurring, and which have been engineered or added to modify the sequence of the antibody.
Recombinant techniques are preferably employed in the production of large quantities of antibodies, antibody fragments and single chain antibodies. Generally, recombinant production of antibodies, antibody fragments or derivatives employs mRNA encoding the desired antibody isolated from hybridoma cells. This mRNA is used to generate a cDNA molecule encoding an antibody or a fragment. Once obtained, this cDNA can be amplified and expressed in eukaryotic and prokaryotic hosts depending on the method.
In some embodiments, derivatives of the antibodies are provided. The term "antibody derivative" as used herein encompasses the antibody itself or a fragment thereof, and may also include redundant moieties. These moieties may increase the water solubility, absorption, biological half-life, etc., decrease antibody toxicity in vivo or in vitro, remove or attenuate adverse side effects of the antibody in vivo, or serve as a marker that allows detection of the presence of the antibody. Structures capable of modulating these effects have been reported in detail. In some cases, labeled, monitorable antibodies are provided. An antibody is said to be "labeled, monitorable" if it binds to a molecule that can be identified, visualized or located by known methods. Suitable detectable labels include radioisotope labels, enzyme labels, nonradioactive isotope labels, fluorescent labels, toxin labels, affinity labels, chemiluminescent labels, and nuclear magnetic resonance contrast agents.
In some embodiments, monoclonal antibodies are provided that are capable of secreting antibodies that selectively recognize particular supramolecular aggregate conformations, such as antibodies that recognize particular pre-fibrillar oligomers, ring fibrils, or amyloid fibrils.
In some embodiments, these monoclonal antibodies can be used to treat or prevent preeclampsia. These antibodies may also be used to qualitatively or quantitatively monitor pre-eclampsia-associated misfolded protein aggregates, such as in a urine sample. These preeclampsia-associated misfolded protein aggregates exhibit specific oligomeric conformations that can be recognized by monoclonal antibodies, and the presence of these conformations can indicate that the patient has developed preeclampsia or is predictive of the risk of the impending preeclampsia syndrome.
SerpinA1 and Albumin as biomarkers for Pre-eclampsia
Applicants have previously demonstrated using proteomics techniques (SELDI-TOF mass spectrometry) in combination with standard molecular and biochemical identification assays that women with preeclampsia have a higher amount of serpin a1 polypeptide or albumin polypeptide in their urine and other body fluids and tissues than women without this disease (u.s.appl.no.:12/084004(PCT/US2006/042585), the contents of which are incorporated herein by reference).
In certain aspects, the invention relates to methods for detecting serpin a1 polypeptide or an albumin polypeptide in a sample (e.g., urine) from a subject to determine the status of preeclampsia. In certain instances, SELDI-based measurements can be used to detect the onset of preeclampsia in a subject. These methods may comprise a step of measuring the levels of up to 13 serpin a1 and albumin polypeptide fragment biomarkers. This method is based in part on the correlation of the appearance of serpin a1 and albumin polypeptide biomarkers with the onset of preeclampsia. 13 serpin a1 and albumin polypeptide biomarkers have been identified (u.s.appl.no.:12/084004(PCT/US2006/042585)), wherein part of the SEQ numbers are listed here:
[MIEQNTKSPLFMGKVVNPTQK(SEQ ID NO:1);
M ox IEQNTKSPLFMGKVVNPTQK(SEQ ID NO:2);
M ox IEQNTKSPLFM ox GKVVNPTQK(SEQ ID NO:3);
EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFS(SEQ ID NO:4);
DAHKSEVAHRFKDLGEENFKALVL(SEQ ID NO:5);
DAHKSEVAHRFKDLGEENFKALVLIA(SEQ ID NO:6)]
as used herein, the term "albumin" refers to the full-length albumin polypeptide as well as a fragment thereof. Examples of albumin polypeptide biomarkers are listed herein as SEQ id no:
[DAHKSEVAHRFKDLGEENFKALVL(SEQ ID NO:5);
DAHKSEVAHRFKDLGEENFKALVLIA(SEQ ID NO:6)]
the full-length amino acid sequence of the wild albumin is Genbank Access No. P02768.
It has been found that samples from pregnant women are indicative of the onset of preeclampsia if the levels of the above 13 biomarkers exceed certain criteria. In these cases, there are two scoring criteria for protein in Urine (UPS): one Boolean score (UPSb), representing the sum of each Boolean indicator assigned to 13 biomarkers, and the other complementary to it is a ranking score (UPSr) that retains the Boolean indicator results for 13 biomarkers with a value of 1 (i.e., objective presence), calculated by: UPSr ═ S/N/10] +1, where S/N is the signal-to-noise ratio in the SELDI analysis. Therefore, theoretically, UPSb scores ranged from 0 to 13 (each serpin a1 and albumin polypeptide biomarker), whereas UPSr could be taken from 0 to infinity. Each score has the most appropriate evaluation criteria for distinguishing between the control group and patients with severe preeclampsia. A UPSb level of greater than 6 and a UPSr level of greater than 8 indicates that the subject has severe preeclampsia. A UPSb level of less than 6 and a UPSr level of less than 8 indicates that the subject does not have severe preeclampsia. If a sample contains other scored combinations of UPSb and UPSr, a second sample should be taken 1, 2, 3, 4, 5, 6, or more days later and this sample can be tested by the methods described herein to determine whether the subject has preeclampsia. Methods for test sample preparation, biomarker detection and measurement, and diagnosis are described herein and in the citation (U.S. appl.no.:12/084004(PCT/US 2006/042585)).
4. Use of VEGF, PIGF and sFlt-1 as biomarkers for pre-eclampsia
Studies have shown that plasma concentrations of Vascular Endothelial Growth Factor (VEGF), placental growth factor (PIGF), and soluble fms-like tyrosine kinase-1 (sFlt-1) vary in women with preeclampsia (Levine RJ et al N Engl J Med2004: 12; 350: 672-83; Maynard SE et al J Clin Invest 2003; 111: 649-58; Levine RJ et al N Engl J Med2004: 12; 350672-83). Applicants have previously demonstrated that VEGF, PIGF and sFlt-1 may serve as biomarkers for the early diagnosis of preeclampsia (U.S. Publ.No: US-2006-0183175; PCT/US2005/047010, the contents of which are incorporated herein by reference). Applicants have demonstrated that urine concentrations of sFlt-1 are significantly elevated and PIGF concentrations are significantly reduced in pregnant women with hypertension. In certain instances, this patent employs a method of measuring PIGF and sFlt-1 in a urine sample and uses the ratio of these two growth factors that act in opposition to each other to distinguish pregnant women with severe preeclampsia from other pregnant women, including individuals who may exhibit mild preeclampsia with or without chronic hypertension, and normotensive controls. The methods described in the patents can also be used to assess the risk of developing certain hypertensive complications in pregnant women, including preeclampsia. These complications may include elevated uric acid levels in blood from caesarean section, elevated systolic and diastolic blood pressure, proteinuria, pregnancy, fetal weight at birth, placental disruption, intra-uterine fetal growth retardation, hemolysis, thrombocytopenia, elevated liver enzymes and HELLP syndrome, among others.
In some cases, an equation is used to analyze the concentration or level of the obtained biomarker. The resulting values can provide the likelihood that a pregnant female will develop hypertensive symptoms, such as preeclampsia. The term "equation" as used herein represents any mathematical expression, algorithm, or other method that can be used to assess the risk of whether the level of a biomarker indicates that a pregnant female has or may have hypertension or complications.
In some cases, this equation may be used to calculate the uFP value for a pregnant female. In this patent, uFP refers to log [ sFlt-1/PIFG × 100 ]. In one aspect, a uFP value of greater than 1.4 is a prognostic indicator that indicates an increased risk of the pregnant woman being treated or protected from hypertension-related syndromes. On the other hand, a uFP value of greater than 2.1 indicates that this pregnant woman is at risk of developing severe preeclampsia. In addition, a uFP value of greater than 2.1 also indicates that this pregnant woman may require caesarean delivery.
Methods for the preparation, detection, biomarker measurement and diagnosis of test samples are described herein and in the citation (U.S. Publ.No: US-2006-0183175).
5. Preparation of test specimens (for some of the methods described in this patent)
The sample may be from a pregnant subject, such as a pregnant woman in need of detection of preeclampsia. The pregnant subject may be a female who has previously been determined to be at high risk of pre-eclampsia based on their personal or family history. A pregnant subject may also have been previously diagnosed as having chronic hypertension. Other subjects may be pregnant women known to have preeclampsia. In some embodiments, the methods described herein can be used to monitor individuals who have been diagnosed with preeclampsia, e.g., to determine the effect of a treatment on a woman with preeclampsia. In addition, the subject may also be a healthy female for routine testing for the presence of preeclampsia, or a baseline level for the subject itself or for other subjects. In other cases, the sample may be from another non-human female pregnant subject, or a non-pregnant subject, such as a method for identifying compounds for treating preeclampsia, and the like.
Urine samples can be tested immediately after sampling or after a period of time, provided that the sample also contains a measurable biomarker. For example, urine samples can be stored at-70 ℃, or mixed and stored in containers pre-treated with reagents capable of stabilizing or preserving the biomarkers. Preferably, the urine sample is taken from the first urine in the morning on an empty stomach.
The biomarkers referred to in this patent may be measured in different biological samples, preferably from liquid samples such as urine. Biomarkers can also be measured in tissue or other biological fluid samples. Examples of other biological samples that can be measured using the methods and kits of this patent include, but are not limited to, blood, plasma, vaginal secretions, cerebrospinal fluid, tears, saliva, and the like. If desired, the sample may be treated to improve the detectability of the biomarkers. For example, a urine sample from a subject can be separated into multiple components. Any method that can enrich for the desired biomarker polypeptide can be used. Depending on the detection method, sample processing, such as a pre-separation step, may be selective and not necessary to improve the detectability of the biomarkers. For example, if an antibody that specifically binds to a biomarker is used to detect the presence of the biomarker in urine, sample processing is not necessary. Sample processing may include separation of the sample and collection of components determined to contain the biomarker. Methods of fractionation include, for example, size exclusion chromatography, ion exchange chromatography, heparin chromatography, affinity chromatography, fractional extraction, gel electrophoresis, liquid chromatography, and the like. Examples of methods of isolation are described in the citation (PCT/US 03/00531).
As an example, the sample may be previously separated by anion exchange chromatography. Anion exchange chromatography can separate proteins in a sample according to charge characteristics. For example, a Q anion exchange resin may be employed and the sample may be subsequently eluted with an eluent of a different pH. Anion exchange chromatography can separate biomolecules that are more negatively charged than other biomolecules in a sample. Proteins eluted with higher pH may have a weaker negative charge and proteins eluted with lower pH may have a stronger negative charge. Thus, in addition to reducing the complexity of the sample, anion exchange chromatography will separate proteins according to binding characteristics.
As another example, biomolecules can also be separated by high resolution electrophoresis, such as one-dimensional or two-dimensional gel electrophoresis. The components containing the biomarkers can be separated and further measured by gas phase ion spectroscopy. A preferred method is to use two-dimensional gel electrophoresis to produce a two-dimensional array of biomolecule spots, which include one or more biomarkers. Examples are given in Jungblout and Thiede, Mass Spectr. Rev.16:145-162 (1997). Two-dimensional gel electrophoresis can be carried out by a commonly used method, see Deutscher ed., Mechors in Enzyniology vol.182. In some cases, biomolecules in a sample can be separated using methods such as isoelectric focusing, where the biomolecules are separated in a pH gradient to a point (isoelectric point) where the respective net charge is 0. This first separation step results in a one-dimensional array of biomolecules, which are then separated by another method, usually different from the first separation step. In a typical method, two-dimensional gel electrophoresis can be used to chemically separate complex biomolecule mixtures with molecular weights of 1000-. The pH of these gels is usually 3-10 (extensive gels).
As another example, High Performance Liquid Chromatography (HPLC) can also be used to separate mixtures of biomolecules in a sample according to physical properties, such as polarity, charge, and size. HPLC equipment typically contains a stationary phase, a pump, a syringe, a separation column, and a detector. A portion of the sample is injected into the column and the biomolecules in the sample are separated by the column. Different biomolecules in the mixture pass through the column at different rates depending on the different partition behavior in the stationary and mobile phases. Components that are the same as the molecular weight or other physical properties of the desired biomarker can be collected. This component can then be used for gas phase ion spectroscopy to detect biomarkers. For example, detection can be by MALDI or SELDI.
Prior to analysis, biomarkers can be selectively modified to increase resolution, or to identify their characteristics. For example, the biomarkers may be subjected to a protein cleavage treatment. Any suitable protease treatment may be employed. Proteases, such as trypsin, often cleave proteins into a specific number of fragments that are very useful as fingerprints for biomarkers that can be used to indirectly determine the biomarkers. This is very useful for distinguishing between biomarkers of close molecular weight. In addition, the cleaved fragments are more suitable for analysis of large molecular weight biomarkers, as small molecular weight biomarkers are easier to analyze with mass spectrometry. Next, the characteristics of the biomarkers can be further analyzed in protein databases using physical, chemical signature matching (e.g., SwissProt).
In some embodiments, a urine sample from a pregnant female may be treated with one or more stabilizing reagents prior to detecting the marker, or the container used to collect the sample may be treated with one or more stabilizing reagents prior to collecting the sample. The term "stabilizer" as used herein refers to one or more molecules, such as polypeptides or nucleotides, which may be used to prevent the degradation of the marker. In some cases, the stabilizer may be a protease inhibitor including AEBSF, Pefabloc SC, antiprotease dihydrochloride, aprotinin, anisole and anisole hydrochloride, bestatin, chymotrypsin inhibitor, E-64, EDTA and its sodium salt, leupeptin, ethylmaleimide, pepstatin and pepstatin A, phosphonodipeptide, sodium azide, trypsin inhibitor, or epsilon-aminocaproic acid.
5. Detection and measurement of biomarkers (for some embodiments described in this patent)
The pre-eclampsia biomarkers used in the present invention can be detected by any of the methods that have been widely used. Examples of such methods include, but are not limited to, immunological methods for detecting secreted proteins, protein purification methods, protein function and activity assays, nucleic acid hybridization methods, nucleic acid reverse transcription methods, and nucleic acid amplification methods. In some cases, the level of the biomarker may be determined by ELISA.
The methods and compositions provided herein allow for the assessment or monitoring of the risk of preeclampsia in a pregnant female by detecting or monitoring the levels of preeclampsia-associated biomarkers (e.g., serpin a1 polypeptide, albumin polypeptide, VEGF polypeptide, sFlt-1 polypeptide, and PIGF polypeptide) in a sample from the pregnant female. This can be accomplished by taking urine samples and measuring the levels of biomarkers at various times after pregnancy using the methods described herein. The values obtained can be compared with the values of a control or with known (predetermined) standards. As used herein, the term "suitable standard" or "control" refers to the level of a biomarker measured in urine obtained from a control individual. Suitable criteria may be determined by urine samples from normal pregnant women and from pregnant women determined to have hypertensive symptoms such as preeclampsia. In some cases, the criteria provided in the present invention are from an individual who is pregnant for the same number of weeks as the test sample was taken. A sample from a subject may be obtained and analyzed simultaneously. Alternatively, preeclampsia-related biomarkers (e.g., serpin a1 polypeptide, albumin polypeptide, VEGF polypeptide, sFlt-1 polypeptide, and PIGF polypeptide) may also be assayed before or after a sample from a subject is used for other routine tests. Standard preeclampsia-related biomarker levels may be obtained by basic skill personnel using methods that are already widely used.
Biomarkers such as serpin a1 polypeptide and albumin polypeptide can preferably be captured using a capture reagent immobilized on a solid support, such as any of the biochips, multi-well microtiter plates, resins, or other suitable supports described herein. A preferred mass spectrometry technique is surface enhanced laser desorption/ionization (SELDI) mass spectrometry, for example, using the method described in U.S. patent No.5,719,060 and No.6,225,047. In these methods, the step of presenting the component to be analyzed to the energy source by the probe on the surface plays an important role in the desorption/instantiation process. In this context, the term "probe" refers to a device that is capable of attaching to a surface containing the probe and presenting the substance to be analyzed to an ionization energy source, ionizing and injecting into a liquid phase ion spectrum (e.g., mass spectrum). A probe typically comprises an immobilised substrate, which may be mobile or immobilised, which substrate comprises a sample presentation surface on which analyte is presented to an ionisation energy source.
In one type of SELDI, referred to as "surface enhanced affinity capture," the probe comprises a chemoselective surface ("SELDI probe"). A "chemoselective surface" binds to an adsorbent, also referred to as a "binding group" or "capture reagent", or a reactive group capable of binding to a capture reagent, for example, by a reaction capable of forming a chemical or coordination bond.
As used herein, a "reactive group" represents a chemical group capable of binding to a capture reagent. Epoxides and carbodiimidizoles are well reactive regions capable of covalently binding to polypeptide capture reagents such as antibodies or cellular receptors. Nitriloacetic acid and iminodiacetic acid are excellent reaction zones capable of coordinating with metal ions that may interact non-covalently with histidine-containing polypeptides. "reactive surface" refers to a surface to which reactive groups are bound. "sorbent" or "capture reagent" refers to any substance capable of binding the desired biomarker. Suitable adsorbents for SELDI in the present invention are described in U.S. patent No.6,225,047.
One type of adsorbent is a "chromatographic adsorbent," which is a material commonly used in chromatography. Chromatographic adsorbents include, for example, ion exchange materials, metal chelators, immobilized metal chelators, hydrophobic interaction adsorbents, hydrophilic interaction adsorbents, dyes, mixed adsorbents (e.g., hydrophobic attractive/electrostatic repulsive adsorbents). "biospecific adsorbents" is another category and refers to adsorbents comprising biomolecules such as nucleotides, nucleic acid molecules, amino acids, polypeptides, monosaccharides, polysaccharides, fatty acids, lipids, steroids or their covalent conjugates (e.g., glycoproteins, lipoproteins, glycolipids). In some cases, the biospecific adsorbent may be a macromolecular structure, such as a protein complex, a biofilm, or a virus. Specific biospecific adsorbents are antibodies, receptor proteins and nucleic acids. Typically, biospecific adsorbents have a higher specificity for the substance being analyzed than chromatographic adsorbents.
Another SELDI, called SEND, utilizes probes that bind energy absorbing molecules to the surface ("SEND probes"). An "energy absorbing molecule" (EAM) herein refers to a molecule that is capable of absorbing energy from a laser desorption ionization source and assisting in the desorption and ionization of a substance being analyzed. EAM comprises molecules for MALDI, representative of which are cinnamic acid derivatives, sinapic acid (SPA), Cyanocinnamic Hydroxide (CHCA) and dihydroxybenzoic acid, ferulic acid, and hydroxyacetophenone derivatives. This category also includes EAMs used in SELDI, which are listed in U.S. patent No.5,719,060.
Another form of SELDI, known as SEPAR, involves the use of a probe that contains a surface-bound region capable of covalently binding to the substance to be analyzed and releasing the molecule to be analyzed by breaking a photosensitive bond after exposure to light, which may be a laser. See U.S. patent No.5,719,060 for an example. SEPAR and other kinds of SELDI can be adapted according to the requirements in this patent for detecting biomarkers or the general condition of biomarkers.
The detection of biomarkers in the present invention may be enhanced with certain selective conditions, such as certain adsorbents and washing solutions. By "wash solution" is meant herein a reagent, usually a solution, which may be used to influence or modify the adsorption of a certain substance to be analyzed on the absorption surface, or to remove unbound substances from the surface. The elution characteristics of the wash solution can be determined by, for example, pH, ionic strength, hydrophobicity, solubilization promoting, detergent strength, and temperature.
In some embodiments of the invention, the sample is analyzed using a "biochip" method. "biochip" refers to a generally planar solid substrate to which is attached a capture reagent (adsorbent). Often, the surface of a biochip comprises a plurality of distinct locations, each of which is immobilized with a capture reagent. The biochip can be used to bind a probe in between, which can then be used for gas phase ion spectroscopy, preferably using mass spectrometry. Alternatively, the biochip can be combined with another substrate to form a probe that can be analyzed spectroscopically.
In the present invention, a number of biochips are available for capturing biomarkers, such as those from commercial sources, e.g., Ciphergen Biosystems (Fremont, CA), Packard Bioscience Company (Meriden, CT), Zyomyx (Hayward, CA), Phytos (Lexington, MA). Ions for these biochips are described in U.S. patent nos.6,225,047,6,329,209 and PCT Publication nos. WO 99/51773 and WO 00/56934.
More specifically, the biochip manufactured by Ciphergen Biosystems comprises a strip-like surface on an aluminium substrate, on which a chromatographic or biospecific adsorbent is located in a position where it can be localized. The surfaces of the strip-shaped structures are coated with silicon dioxide.
CiphergenTypical of the arrays are H4, SAX-2, WCX-2 and IMAC-3 biochips comprising functionalized cross-linked polymers. These molecules are either physically attached to the biochip surface by way of water condensation, or covalently attached to the biochip surface by way of silanes. The H4 biochip has isopropyl properties for hydrophobic attachment. SAX-2 biochips have quaternary ammonium properties for anion exchange. The WCX-2 biochip has carboxylic acid properties for cation exchange. IMAC-3 biochips have nitriloacetic acid properties for the uptake of transition metal ions in coordinated form, such as Cu + + and Ni + +, which in turn can be used for the coordinated adsorption of biomarker molecules.
The substrate containing the adsorbent is contacted with the urine sample for a sufficient period of time to allow binding of the biomarker, if present, to the adsorbent. After a period of incubation, the substrate surface is washed to remove unbound material. Any suitable detergent may be used, preferably a liquid solvent. Next, the energy absorbing molecule is used to interact with the substrate to which the biomarker is bound. Energy-absorbing molecules are molecules that absorb energy from an energy source in the gas phase ion spectrum, which assist in desorption of the biomarker from the substrate. Exemplary energy absorbing molecules include cinnamic acid derivatives, sinapinic acid, and dihydroxybenzoic acid. A preferred energy absorbing molecule is sinapic acid.
Once adsorbed onto a substrate, such as a biochip or antibody, any suitable method can be used to measure the biomarkers in the sample. For example, biomarkers can be determined by a number of detection and measurement methods, such as gas phase ion spectroscopy-like methods, optical methods, electrochemical methods, atomic force microscopy and radio frequency methods. These methods can be used to detect one or more biomarkers.
In some cases, the detection and measurement of biomarkers is by mass spectrometry, in particular by SELDI. SELDI refers to a method of desorption/ionization of gas phase ion spectra (e.g., mass spectrometry) where the species to be detected is captured on a surface containing a SELDI probe, in SELDI MS the gas phase ion spectra used are mass spectra. SELDI technology is described in more detail above.
In other cases, the biomarker may be detected in the sample by immunoassay. Immunodetection methods employ antibodies to specifically bind antigens (e.g., biomarkers). Immunoassay is characterized by the use of the specific binding of antibodies to antigens for the isolation, binding and quantitative measurement of certain antigens. Thus, under a given immunoassay condition, an antibody binds to a particular antigen more than 2 times as well as to background and does not bind significantly to other proteins in the sample. Such specific binding may require that the antibody be specifically screened for a particular protein. For example, polyclonal antibodies prepared using a biomarker molecule from an animal such as rat, mouse or human can be screened to obtain polyclonal antibodies that react only with this biomarker from this species and not with other proteins, which do not include the product of the genetic polymorphism and the product of different alleles of this biomarker. This screening can be accomplished by removing the portion of the antibody that cross-reacts with this protein from other species.
Antibodies (e.g., serpin a1 polypeptide or albumin polypeptide) that specifically bind to a biomarker can be prepared using purified biomarkers or their nucleotide sequences in a known manner. Methods can be found in Coligan, Current Protocols in Immunology (1991); harlow & Lane, Antibodies A Laboratory Manual (1988); goding, Monoclonal antibodies: Principles and Practice (2d ed. 1986); kohler & Milstein, Nature 256: 495-; huse et al, Science 246: 1275-; ward et al, Nature 341:544-546(1989).
Typically, a sample from a subject may be reacted with an antibody that specifically binds to a certain biomarker. The antibody may also optionally be bound to a solid support to aid in washing and separation of the complex prior to binding the antibody to the sample. The solid support is of glass or plastic quality and is in the form of a microtiter plate, rod, bead or microsphere, etc. The antibody may also be attached to a probe substrate, or protein chip.
After co-incubation of the sample and the antibody, the mixture is washed and the resulting antibody-biomarker complex is detected. This can be achieved by incubating the washed mixture with a detection reagent. Such a detection reagent may be, for example, a secondary antibody to which a detectable label is attached, or the like. Typical detectable labels include magnetic beads, fluorescent dyes, radioactive labels, enzymes (such as horseradish peroxidase, alkaline phosphatase, and the like commonly used in ELISA), and colored labels such as colloidal gold or colored glass or plastic beads. Alternatively, the biomarker in the sample may be detected indirectly, for example by using a secondary antibody to which a label has been added to detect antibodies that specifically bind to the biomarker, or by using a competitive or inhibitory detection method, for example by incubating the previous mixture with a monoclonal antibody that binds to a particular epitope of the biomarker to be detected.
Methods for detecting the presence and amount of antibody-biomarker complexes include, for example, detection of fluorescence, luminescence, chemiluminescence, absorption, reflection, emission, birefringence, refractive index (e.g., surface plasmon resonance, ellipsometry, resonance mirror methods, gated waveguide coupler methods, or interferometry), and the like. Optical methods include microscopy (confocal and non-confocal), imaging methods, and non-imaging methods. The emission frequency method includes multipole resonance spectroscopy. Many practical methods have been widely adopted, including for example: enzyme linked immunosorbent assays (EIAs) such as enzyme linked immunosorbent assays, radioimmunoassays, Western blot assays or slot blot assays.
The immunoassay method can either determine the presence or absence of a biomarker in a sample or determine the amount present. The amount of antibody-label complex can be determined by comparison with a standard. The standard may be, for example, a known compound or the amount of other proteins in the sample. It will be appreciated that the amount of biomarker is not measured in absolute terms, but rather is expressed as a ratio to a standard.
When data is generated after sample measurement, for example, after measurement by mass spectrometry, the data can be analyzed by computer software. In some cases, biomarkers bound to a substrate can be detected in gas phase sample spectra, the biomarkers ionized by an ion source, such as a laser, and the resulting ions collected by ion optics and then separated and analyzed in a mass analyzer. The detector then translates the information of the detected ions into a form of mass-to-charge ratio. Thus, both the content and the quality of the biomarker can be detected.
Generally, the data from the desorption and detection of the biomarkers can be analyzed by an electronic computer. The computer program analyzes the resulting data to indicate the number of biomarkers detected and selectively displays the signal intensity and molecular weight of each biomarker. The data analysis may also include determining the signal intensity of the biomarkers and removing data that deviates from a pre-set data profile. For example, the observed peaks may be normalized by calculating the intensity of each peak relative to some reference value. The reference value may be the background noise generated by the machine or chemical agent, such as the energy absorbing molecules themselves, and this signal is set to a zero value at the time of analysis.
The computer can convert the data into different display formats. Standard spectra can be used for display, but a more practical approach is to display only peak height and quality information, which results in a cleaner image and makes biomarkers of close molecular weight easier to distinguish; another practical approach is to compare one or more spectra and label the up-or down-regulated biomarker molecules between different samples. Using these methods, it is possible to determine whether a particular biomarker is present in a sample.
The software for analyzing the data may include an algorithm to analyze whether a signal peak represents a biomarker. The software may also be used to apply the data of observed biomarker peaks to classification tree or artificial neural network analysis to determine whether a biomarker peak or combination of peaks represents a diagnosis of intra-amniotic inflammation.
7. Multiple or Integrated analysis for preeclampsia
In some embodiments, the invention provides methods for detecting or predicting preeclampsia that may employ one or more methods for measuring one or more preeclampsia biomarkers, e.g., by various methods, and the subsequent measurements may be used to confirm the results of the previous test, or to obtain more qualitative and quantitative results, which may improve the predictive value of the results and provide greater confidence in the diagnosis. For example, a method of Congo Red Retention (Congo Red Retention) testing (e.g., dot blot) may be initially used to obtain a first indicator as to whether a subject has or is likely to develop preeclampsia. This first step of testing can be done by the subject himself without the need for monitoring or laboratory equipment, or with only minimal skill and experimental equipment.
Further quantification may be performed if the results of the congo red retention test are positive, an indicator that may also be used to detect or predict the severity of pre-eclampsia. In addition to the congo red retention test and similar dye-based tests, the samples can also be used for antibody-based tests, for example using antibodies that detect a particular conformation of misfolded protein aggregates. These tests may employ the same sample as used for the congo red retention test, or a second sample from the same subject; the test may be performed after the congo red retention test, or may be performed simultaneously. These tests using antibodies that recognize specific conformations of misfolded protein aggregates may be used in some embodiments to confirm the results of congo red retention tests, or may be used to obtain more qualitative information, such as correlating the presence or absence of a particular protein oligomer conformation with the likelihood of developing preeclampsia, or indicating the severity of preeclampsia. In addition to these pre-eclampsia sample protein aggregate-based tests (e.g., congophilic/congo red retention tests and antibody-based protein aggregate analysis), other tests may be used to confirm the results of these tests, or to obtain more quantitative and qualitative data. For example, additional tests may be used to detect the presence of additional biomarkers, including the preeclamptic biomarkers albumin, SerpinA1, sFlt-1, PIGF, VEGF, by ELISA or by SELDI-TOF analysis of protein fragments. In addition, ratios of predictive biomarker levels, such as sFlt/PIGF [ uFP ], may be calculated. Further, the presence or relative levels of other proteins may also be used in the diagnosis of pre-eclampsia, such as immunoglobulins in urine protein aggregates, ceruloplasmin, etc.
It should be noted that the test methods described herein may be performed in any order, either individually or in any combination. The invention is not limited in this respect. Applicants have also previously demonstrated and described that any of these test methods is sufficient to detect preeclampsia, and aid in diagnosis and prognosis.
8. Treatment of
Protein conformation diseases, such as senile dementia, light chain amyloidosis and prion diseases, are exacerbated by the growth and aggregation of amyloid fibrils, due to misfolding of intracellular proteins into abnormal 3D structures. Recent observations have shown that soluble pre-amyloid oligomers (intermediates of fibrous tissue) are protein toxic and can lead to epidermal destruction and oxidative stress. Applicants have demonstrated that epidermal breakdown and oxidative stress play a pathogenic role in the pre-eclampsia period. Applicants have unexpectedly discovered that preeclampsia is a conformational disease and is associated with protein misfolding and aggregation, characterized by the aggregation of amyloid proteins. Applicants have further demonstrated, using conformational specific polyclonal or monoclonal antibody staining methods, that misfolded intermediates in the urine of subjects with preeclampsia have a tendency to assemble into pore-like structures (amyloid channels), which may play a significant role in the course of clinical pathogenesis, such as epidermal destruction and oxidative stress. Applicants conclude that the accumulation of abnormal protein aggregates in the placenta of patients diagnosed with preeclampsia indicates that such protein aggregates are one of the causative factors of the disease. In some embodiments, methods are provided for treating preeclampsia, including immunological and pharmacological strategies, and based on blocking the production of misfolded protein oligomers that may assemble into amyloid channels. In some embodiments, methods of treating preeclampsia are provided. In some embodiments, these methods of treatment are based on methods that have been found to reduce the severity of other protein misfolding diseases (e.g., senile dementia). In the course of studying new therapeutic approaches for diseases like abnormal protein aggregation, such as senile dementia and other amyloidosis, new drugs have been discovered that inhibit the formation of new protein aggregates or reduce the burden of existing protein aggregates. Our findings relating to the association of preeclampsia with abnormal aggregation of proteins indicate that preeclampsia can be treated with these drugs.
Various methods and drugs have been developed and used to treat Alzheimer's disease by inhibiting or reducing amyloid-beta aggregation in Alzheimer's disease, such as small molecule inhibitors, peptide inhibitors directed against fiber or oligomer production, immunization against one or more protein aggregate components, passive antibody immunization with antibodies or antibody fragments directed against one or more protein aggregate components. Some of these agents have been shown to inhibit aggregation of proteins other than beta-amyloid. For example, p-aminophenol and 2-amino-4-chlorophenol and their derivatives (Cell Biochemistry and Biophysics 44:549-553(2006)) are capable of inhibiting the aggregation of other proteins.
In some embodiments, the invention provides methods of treating preeclampsia using a protein aggregation inhibitor to inhibit the formation of protein aggregates, or to reverse protein aggregates that have formed. In some embodiments, agents that can stabilize the conformation of the original aggregation-prone protein are provided to reduce the rate of aberrant folding and subsequent aggregation. In some embodiments, small molecule inhibitors, peptide inhibitors, directed to fiber or oligomer production, immunization of one or more protein aggregate components, passive antibody immunization with antibodies or antibody fragments directed against one or more protein aggregate components are provided as a method of treating or preventing pre-eclampsia. In some embodiments, anti-beta amyloid agents (as summarized in Drug Discovery data: therapeutic strategies 1:7-12 (2004)) are provided to treat or prevent pre-eclampsia.
Applicants have also found that serpin a1 or a polypeptide fragment thereof is one of the components of preeclamptic protein aggregates. serpinA1 has been reported in the past as a biomarker for early diagnosis of preeclampsia (U.S. appl.No.:12/084004(PCT/US 2006/042585)).
The term "serpin a1 polypeptide" as used herein refers to the full-length serpin a1 polypeptide and also to a fragment of the full-length serpin a1 polypeptide. serpinA1 has been identified in the past as a serpin, and an alpha 1 antitrypsin. The Genbank sequence of the SerpinA1 polypeptide is No. P01009. It is understood that the serpin A1 polypeptide encoded by another serpin A1 allele may also be used to detect whether a subject has preeclampsia. For example, serpin a1 polypeptides encoded by the M1A, M2, or M3 alleles can be used to diagnose or assess preeclampsia in a subject. Serpin a1 polypeptide was synthesized in liver and embryonic trophoblasts and exists in a variety of forms unrelated to the anti-proteolytic effect of serpin a 1. Serpin a1 polypeptide was highly susceptible to oxidation, and strong oxidative stress caused oxidation of serpin a 1. In vitro, a fragment of the polypeptide from the C-terminus of the full-length SerpinA1 polypeptide was able to elicit oxidative responses and neutrophilic chemotactic behavior.
Identification of SerpinA1 in the urine of preeclamptic patients indicates that preeclampsia has a similar etiology to other diseases such as alpha-1 antitrypsin deficiency, which is caused by accumulation of misfolded alpha-1 antitrypsin leading to hepatocyte damage and cirrhosis (N.Engl.J.Med.346:45-53 (2002); J.Clin.Inv.110:1585-1590 (2002)). In some cases, therapeutic agents capable of preventing abnormal aggregation of serpin a1 protein or a fragment thereof are provided. In other cases, reagents are provided that are capable of disaggregating existing aggregates of serpin a1 or fragments thereof. For example, one such reagent is trimethylamine nitroxide and related compounds (am.J.Respir.cell mol.biol.24:727-732 (2001)). Another example is the FLEAIG polypeptide (SEQ ID NO:7) and related polypeptides and derivatives (am.J.Respir.cell mol.biol.35:540-548 (2006)). In certain instances, trimethylamine nitroxide and related compounds, or FLEAIG polypeptides (SEQ ID NO:7) and related polypeptides and derivatives are provided as a useful therapeutic to treat or prevent preeclampsia.
As will be appreciated by those of ordinary skill in the art, the assessment of a treatment regimen may also be based on the assessment of the preeclampsia syndrome and the end result. Thus, the invention may also be used to determine the onset, progression or remission of a subject as represented by one or more biomarkers (e.g., serpin a1, albumin, sFlt-1, PIGF, VEGF, misfolded protein aggregates/congophilic red substance). In some embodiments, the methods of the invention can be used to detect the level of one or more biomarkers in a subject who has been diagnosed with preeclampsia. In other embodiments, the methods of the invention can be used to obtain measurements useful for diagnosing preeclampsia in an individual. In some embodiments, the subject may already be under treatment with a drug for preeclampsia, while in other embodiments the subject may not have been treated for preeclampsia.
The method of selecting a treatment for preeclampsia may be based in part on the presence of abnormally high levels of preeclampsia biomarkers (e.g., serpin a1, albumin, sFlt-1, PIGF, VEGF) or misfolded protein aggregates associated with preeclampsia and exhibiting congophilic properties in the pregnant female. Methods of treatment may include the use of a particular class of drug, a change in mode of action, or a change in diet, based at least in part on the presence of a particular preeclampsia indicator (e.g., detection of one or more biomarkers, congophilic/misfolded protein aggregates, or a particular protein oligomer conformation). These individuals may already be receiving drug treatment for preeclampsia. According to the present invention, the treatment regimen of a subject may be adjusted based on the detection of one or more biomarkers, congo red/misfolded protein aggregates, or a particular conformation of protein oligomers. An individual who may not have previously received any treatment for preeclampsia may be monitored for one or more biomarkers, congophilic/misfolded protein aggregates, or a particular protein oligomer conformation, which may help to select the most effective treatment regimen.
9. Reagent kit
In some embodiments, the invention provides kits that can be used in the described methods. The reagents contained in the kit may include labeled compounds or reagents capable of detecting congophilic or misfolded protein aggregates in urine. In some embodiments, the kit may further comprise a support surface having an affinity for proteins, which may be contacted with a sample (e.g., a urine sample); the kit may further comprise a dye (e.g., congo red) capable of binding to the misfolded protein aggregates associated with preeclampsia, the dye being adapted to contact the sample; the kit may also contain a vessel, tube or container for mixing the sample with a dye or for incubating a protein-avid support surface bound to a protein in the sample. The kit may also include one or more washing solutions for removing dye that is not bound to misfolded protein aggregates. The kit may further comprise positive and negative controls to help control dye binding and washing conditions and times to ensure adequate binding and washing on, for example, a support surface ("test strip") having an affinity for proteins as described above.
In some embodiments, the invention provides kits suitable for use in the detection methods of the invention, where the reagents contained in the kits can be labeled compounds or methods capable of detecting misfolded protein aggregates in urine and the amount thereof (e.g., providing antibodies capable of binding to aggregates), and suitable reagents capable of binding to biomarkers include antibodies, antibody derivatives, antibody fragments, and the like. For antibody-based kits, such kits may comprise, for example: (1) first antibody: one antibody for binding to the biomarker (e.g., bound to a solid support), (2) a second antibody: an antibody, different from the first antibody, that binds to the first antibody or the biomarker and carries a detectable label.
Pre-eclampsia associated biomarkers that may be detected using this kit include sFlt-1, PIGF, VEGF, albumin, SerpinA1, IFI6, and ceruloplasmin. In some embodiments, the kit contains antibodies that specifically recognize certain misfolded protein conformations (e.g., fiber conformations), and the like. The antibody may bind to a particular protein aggregate conformation in a sample of a type detectable by the kit. In some cases, the sample is a urine sample containing pre-eclampsia-associated protein aggregates that contain immunoglobulins, and the secondary antibodies may be pre-adsorbed with Ig to reduce or prevent non-specific binding of the secondary antibodies to the immunoglobulin-containing protein aggregates.
The kits, in some embodiments, may also contain other components, such as a buffer, a preservative, or a protein stabilizer. The kit may also comprise another component for detecting the detectable label (e.g., an enzyme or a substrate).
Each component of the kit may be contained in a separate container, and all of the various containers may be contained in a single package, including instructions for interpreting the results of the assay. The container may be pretreated with a stabilizer, which may also be a component of the kit.
Examples
Examples1
Method
Dot blot of urine sample Congo red dot test procedure
1. The collected urine samples were centrifuged. 1500g, 15 min, 4 deg.C
2. Protein concentration in urine samples after centrifugation was measured using Pierce BCA kit (Thermo Scientific Cat # 23225). To measure protein, urine samples may need to be diluted 12-fold. Urine samples obtained from women potentially at risk of preeclampsia vary widely in protein concentration and in healthy human urine in terms of in vivo water content. Therefore, it is desirable to be able to equalize the total protein concentration. Of more than 680 female urine samples, only about 5% (37/681) of total protein was less than 2 mg/mL. About 60% of the urine samples contained more than 6.6mg/mL total protein. Overall, there is in most cases no need to dry the sample, e.g. using SpeedVac. The drying method has been examined and the results demonstrate that drying does not alter the red dot blot results of the samples. To maintain consistency, SpeedVac drying was used in all tests.
3. A volume of sample was added to the conical centrifuge tube to contain 200. mu.g of protein. Centrifuge to dryness using SpeedVac.
4. Congo red stock solutions (5mg/ml) were prepared. Congo Red (Sigma, Cat # C6277) was dissolved in water. The undissolved powder was separated by centrifugation at 14,000g for 10 minutes. The stock solution should be freshly prepared and should be used within one hour of preparation (e.g., adding a urine sample). Alternatively, Congo red working solution (0.1mg/ml) was prepared by diluting the stock solution 50 times.
5. A blank sample was prepared by adding 2. mu.l Congo Red stock to 0.1mL PBS.
6. Taking the dried urine sample off the SpeedVac, adding 30 mu l of Congo red working concentration solution, and shaking and mixing uniformly.
7. The sample was shaken vigorously for 1 hour using a shaker to bind congo red to the sample.
8. After one hour, dot-blot on nitrocellulose membrane (0.2 micron pore size) with 5 μ l of sample mixed with congo red. Each spot should contain 33.3. mu.g of protein. While a blank sample is spotted. If more than one membrane is used, blank sample spots are required on each membrane. Preferably, a positive control is added for each test. The grid is added in front of the transilluminator, and the nitrocellulose membrane is placed on the transilluminator for spotting, so that each spot can be distributed regularly, and the locus of each sample can be tracked.
9. The film was allowed to dry for 15 minutes.
10. Washing in water for 3 minutes
11. Photograph (all spots should have similar staining intensity at this time)
12. Wash in 50% methanol for 3 minutes, 70% methanol for 1 minute, and finally 90% methanol until the red color in the blank disappears (typically 7 to 10 minutes).
13. Photographs were taken (intensity of staining of each spot, e.g., positive control and blank sample, should appear different).
14. The images were scanned before and after methanol washing, respectively, and the Optical Density (OD) of each point was calculated. Any density measurement software may be used, such as NIH Image J.
15. Percent congo red binding (CRI) was calculated for all samples by: (average OD value before methanol washing of the same sample/average OD value of blank sample on the same piece of membrane) X100.
16. The percentage Congo Red Retention (CRR) was calculated for all samples, including the blank sample. The calculation method comprises the following steps: (average OD value after methanol wash of same sample/average OD value before methanol wash of same sample) X100; subsequently, the CRR of the blank sample was subtracted from all urine sample spot values. If the methanol wash is complete, the spots of the blank sample should not have any residual red color and the OD mean should be 0.
17. Red spots with CRR > 20% were designated as test positive, those with CRR < 15% were designated as negative.
18. If the CRR is between 15% and 20%, the test is repeated. According to the test results, the diagnostic and prognostic diagnostic boundaries of 223 samples analyzed were approximately 16.1%.
Care should be taken to bring all incubation and preparation (e.g. centrifugation) times, volumes, concentrations close to optimal values. However, different times, volumes and concentrations may give reliable test results and may be determined by not too many experiments, and thus the above incubation and preparation times, volumes and concentrations are not limiting, and further, all the possibilities described herein for using chemicals and/or instruments are not particularly limited and other equivalent compounds and/or instruments may be used. In addition, it is noted that some steps are optional, the order between the steps may be changed, and some steps may be deleted or combined. Such variations would be obvious to one having ordinary skill. For example, incubation times of less than 1 hour (step 7), addition of non-denaturing solvents (e.g., Tween20) to the urine sample, or washing with ethanol, isopropanol, instead of methanol, all give reliable test results.
Research design:
the enrolled 110 women were divided into 3 groups according to the preset settings: a normotensive control group (CRL n: 49, GA: 28 [ 21-34 ] weeks); chronic hypertension (cHTN 12, GA 29 [ 24-34 ] weeks) and severe preeclampsia (sPE n 49, GA 30 [ 24-34 ] weeks). In addition, 34 women during pregnancy which were asymptomatic but belonging to the high risk group of preeclampsia were followed for a long period of time. Urine Congo Red staining was quantified by dot blot fixation and spectral shift assay (spectral shift assay) using equal amounts of protein. The stained proteins were identified by tandem mass spectrometry and verified by Western blot. Urine proteomic fingerprints were generated using SELDI-TOF. Placental protein congo red staining was detected by polarising microscopy.
Results
FIG. 1 shows photographs of a nitrocellulose membrane for Congo red dot detection before (left) and after (right) washing (amount of dots on the nitrocellulose membrane is 5. mu.l/dot). As shown in the figure, Congo Red stains consistently from each sample before washing, but post-wash staining was only for urine samples from women with severe preeclampsia (blank samples are negative controls). FIG. 2 shows a photograph of a set of laterally compared urine samples (24 specimens) before and after washing after the Congo Red dot test (33 μ L/dot on nitrocellulose membrane). The boxed numerical samples were from women diagnosed with severe preeclampsia (sPE). As shown in the figure, Congo Red stains each sample similarly before washing, but only urine samples from women with severe preeclampsia after washing. FIG. 3 shows a photograph of a set of longitudinally compared urine samples (28 specimens) before and after washing after the Congo Red dot test (33 μ L/dot on nitrocellulose membrane). Six pregnant women are tracked in the whole course of the whole pregnancy process, and urine is extracted for a plurality of times for analysis. Patients 2 and 5 later developed preeclampsia. Patient 5 samples with boxes were taken at clinically confirmed disease stage. Samples U348i and U348j were samples taken after medically indicated delivery (emergency caesarean section) due to preeclampsia. As shown herein, two patients already had a positive result in an abnormal congo red spot urine sample (red spot positive) as early as before they were diagnosed with preeclampsia. FIG. 14 shows a graph predicting medically indicated delivery due to preeclampsia in longitudinally tracked patients. 36 women at high risk (n-30) or low risk (n-6) were tested by congo red test. Wherein four of the individuals of this group had severe preeclampsia and had a medically indicated delivery. The data herein show that a woman with pre-eclampsia has a high urine congophilic red in the first test sample. We were unable to take an earlier sample to confirm whether these women had such a positive result prior to pregnancy. Fig. 4 is a bar graph showing the congo red retention value (CRR) for pregnant women with severe pre-eclampsia (sPE), chronic hypertension (cHTN), and normal pregnancy (CRL). Wherein congo red retention values are normalized to total protein concentration. As shown, CRR is clearly associated with and has a good indicator of severe preeclampsia. The urine samples were tested at the time of admission after the patient was diagnosed with severe eclampsia. FIG. 5 shows ROC curves for Congo red retention and Congo red binding coefficients for several groups: A) confirmed preeclampsia, B) predicted a medically indicated delivery (223 urine samples taken from 114 different pregnant women) due to preeclampsia, and several women had serial urine samples analyzed during pregnancy. Figure 6 shows that congo red retention of urine proteins correlates closely with the presence and severity of preeclampsia, as analyzed using an abnormal proteomics map (UPSr).
Figure 7 shows that the congo red retention value of urine protein correlates with the ratio value uFP, uFP: log [ sFIt-1/PIGF x 100], as here and U.S. Publ.No. US-2006-0183175; as described in PCT/US 2005/047010. FIG. 8 shows that similar to misfolded proteins found in other diseases (e.g., Alzheimer's disease and Raney virus disease), misfolded proteins found in pre-eclamptic urine can also be isolated in the same manner: such as (a) dot blotting using congo red affinity (samples in boxes from women clinically diagnosed with severe preeclampsia, as described above) (B) gel filtration, or (C) centrifugation, and washing the precipitated congo red binding protein with water (e.g., for (B) and (C)).
In further experiments, placental tissue sections were stained with congo red. Cloud-like birefringent material build-up in syncytropophoblasts occurred in pre-eclamptic sections, whereas none of the control compositions. Figure 15A shows placental tissue sections from three women, two of whom were born preterm due to severe preeclampsia (a-F), and the other was born artificially sudden preterm (control, G-H). These sections were stained with congo red and then viewed under a microscope using white light (A, D, G) or polarized light (B, C, E, F, H and I), with further magnification (640x) of the squares in groups C and F, respectively, in groups B and E. As shown in B and E, the placenta of women with preeclampsia showed cloudy grayish blue (B and C) or grayish green (E and F) birefringent substances accumulated in the syncytrophoblast layer (red arrows). Group I: polarized light images of brain sections from alzheimer's patients were stained with congo red under the same conditions.
FIG. 15B shows images of sediments with a positive response to Congo Red in urine samples from preeclamptic patients. After binding to congo red and centrifugation, the pellet (a) is washed three times with water (repeated centrifugation between washes), resuspended with a drop of water, and then placed on a slide for observation using microscope polarized light (B), or placed on a grid of an electron microscope after staining with 1% uranium acetate (uranyl acetate), and the staining can be (C) positive or (D) negative. The arrows point to a non-cellular structure, most likely a fibril.
In further experiments, congo red stained material was electrophoresed on SDS gel along with those bands that were immunologically active against conformational antibodies and analyzed by mass spectrometry. Fragments of antitrypsin, ceruloplasmin, heavy and light chains of IgG were detected in both urine proteins and in bands immunologically active against conformational antibodies in pre-eclamptic patients. Western blot assays for SERPINA1 were performed on congophilic substances and a number of graded fragments were observed that were capable of binding to antibody responses. Western blot or dot blot tests also showed that patients pre-eclampsia contained ceruloplasmin and IFI6-16 in their urine. ELISA verified the presence of light chain. FIG. 16 shows urine samples (U647 and U648) taken from two patients with severe preeclampsia, loaded on a double 4-20% reducing SDS PAGE gel, partially enriched for misfolded proteins by Congo Red affinity precipitation ( lanes 3, 4 and 7, 8, red asterisks), and partially not enriched ( lanes 1, 2 and 5, 6). The left panel shows the total protein content of the gel after Coomassie blue staining (lanes 1-4). In the samples after congo red precipitation, many new bands became visible. The right panel shows the immunological activity of the protein transferred onto nitrocellulose membrane against serpin A1(SERPINA 1). Additional ladder segment patterns are visible in lanes 7, 8, but not in lanes 5, 6. Each group has molecular weight markers.
This also suggests that congo red can be used to efficiently concentrate misfolded and aggregated proteins in a biological sample (here, a urine sample from a preeclamptic patient) for further study.
In summary, the data show that pre-eclampsia is characterized by a significant increase in congophilic protein in urine samples compared to CRL and cHTN, independent of GA (P < 0.001). In the longitudinal long-term observation group, congophilic proteins appear in urine of women (n-4) who were born prematurely due to severe preeclampsia already 8-10 weeks before clinical symptoms. Congo Red binds to proteins in pre-eclamptic urine samples, producing a red-shift in light absorption, similar to that observed in other amyloid supramolecular protein structures. The urine congo red affinity results correlated with the pre-eclamptic proteomic library characteristics (r 0.89, P < 0.001).
The above data demonstrate that preeclampsia is a pregnancy disease marked by amyloid supramolecular protein aggregates and congo-red protein in urine. A patient with preeclampsia can be detected by identifying congo red-philic protein in urine by dot blot and spectral shift tests, and a diagnostic method is provided. The urine congophilic protein aggregate detected by the method can diagnose the existing preeclampsia and can also predict whether the existing preeclampsia will develop into the preeclampsia in the future (clinical result). As a supplement to the detection of congo red-philic protein aggregates in urine, the congo red staining results of placental tissue samples also showed the hallmark characteristics of such protein aggregates. The method of using congo red staining in placental tissue can also be applied in the diagnosis of pre-eclampsia.
It should be understood that, in addition to congo red, a variety of other chemical reagents and methods known in the art can be used to detect analytical protein aggregates (e.g., thioriboflavin).
Example 2
Method
Research and design:
the 347 women recruited in advance are grouped according to the preset group: normotensive control group (CRL n 98, GA: 27 [ 7-42 ] weeks); chronic hypertension group (cHTN n 40, GA: 32 [ 11-41 ] weeks); gestational hypertension group (gHTN ═ 8 GA: 37 [ 26-39 ] weeks); the mild preeclampsia group (mPE n ═ 36, GA: 36 [ 24-41 weeks ]); the severe preeclampsia group (sPE n 117, GA: 32 [ 22-42 ] weeks) and the superimposed preeclampsia (spPE n 33 GA: 33 [ 18-40 weeks'). In addition, 35 asymptomatic pregnant women were long-term follow-up observed throughout pregnancy and a series of tests were performed. The congo red spot test was standardized by isocratizing urine protein and objectively quantified by percent congo red retention over a few minutes. CRR assesses the predictive power of an indicated delivery (IND) due to preeclampsia by comparison with the protein-creatinine ratio (P/C) as described herein and (U.S. Publ.No.: US-2006-0183175; PCT/US 2005/047010).
Results
Preeclampsia is characterized by increased secretion of misfolded proteins with affinity for the azo dye Congo Red (CR). Congo red dye is also used to detect abnormal amyloid aggregates in Alzheimer's disease and prion disease. The congo red spot test CRR was designed and demonstrated to be a urine congo red protein-based method of pre-and prognostic diagnosis of preeclampsia based on the detection of the overall or total amount of misfolded proteins in a urine sample during pregnancy. FIG. 9A shows CRR values for urine proteins of different experimental groups (CRL: control; cHTN: chronic hypertension; gHTN: gestational hypertension; mPE: mild pre-eclampsia; sPE: severe pre-eclampsia; spPE: superimposed pre-eclampsia). 61% (211/347) of the women had an IND: CRL: 4 percent; crHTN: 40 percent; gHTN: 75 percent; mPE: 69%; sPE: 99 percent; spPE: 100 percent. 77% (162/211) of IND women were born preterm, 51% (107/211) for less than 34 weeks. In mPE, CRR increased somewhat, and in sPE and spPE the increase was more pronounced, independent of GA values. Women with indicated births had elevated CRR upon addition to the study (figure 9B, P < 0.001). Fig. 9C shows that 11% (4/35) of women observed by long-term follow-up had an indication of preterm birth (IND). In this group, CRR began to rise 14 ± 4 weeks before clinical diagnosis of preeclampsia symptoms. CRR predicted more accurately for IND compared to urine samples sFlt1/PlGF (P ═ 0.014) and urine protein/creatinine ratio (P/C) (fig. 9C).
In summary, the determination of global protein misfolding by CRR is a simple method for pre-eclampsia diagnosis and prediction of IND, which is one of the major causes for preterm birth.
Example 3
Method
Research and design:
urine samples from 111 women enrolled were divided into 3 groups: severe preeclampsia group (n 49, GA: 28 ± 1 weeks); chronic hypertension group (cHTN n-12, GA: 29 ± 1 week) and normotensive control group (CRL n-50, GA: 28 ± 1 week). Equal amounts of urine protein were used for dot blot detection. Three conformation-specific antibodies are used here, recognizing the precursor fibre-soluble oligomeric chains (a11, Invitrogen), the ring fibrils (Officer) or the fibres (OC), respectively. Specificity can be verified by deleting the primary antibody. The components of aggregate proteins were identified by mass spectrometry and verified using Western hybridization and sequence-specific antibodies.
Results
Diseases caused by abnormal protein conformation, such as alzheimer's disease, light chain amyloidosis, and raney virus disease, are exacerbated by the formation and aggregation of amyloid fibrils. This aggregate is caused by misfolding of cellular proteins into an abnormal 3D result. Soluble pro-amyloid oligomeric chains (intermediates of fibrillar aggregation) have a proteolytic effect, leading to endothelial damage and oxidative stress.
Some important features in the pathology of preeclampsia are activation of the cardiovascular endothelium, and subsequent vasospasm. Theories as to their cause include abnormal implantation and development of the placenta, oxidative stress, abnormal endothelial prostaglandin and nitric oxide balance, genetic diversity, abnormal autoantibody circulation, and abnormal maternal systemic inflammatory response (Buhimschi IA et al, Hum Reprod Update 1998; 4: 25-42; Ward K et al, Nat Genet 1993; 4: 59; Wallukat G et al, J Clin Invest 1999; 103: 945) 952; fas MM et al, Am J Obstet Gynecol 1994; 171: 158-64; Robert JM 1993; Lancet JM 341: 1447-51). Given that endothelial injury and oxidative stress play a pathogenic role in the severe preeclampsia, the relationship between soluble precursor amyloid oligopeptides in urine and preeclampsia was characterized and classified.
Antibodies used to detect urine soluble precursor amyloid oligomers can detect proteins in several conformations including: i) precursor fiber soluble oligomeric conformations, such as the "a 11" antibody and the like. ii) a ring-fibril conformation, such as an "Officer" antibody or the like, and iii) a fiber conformation, such as an "OC" antibody or the like.
Urine spot hybridization including urine protein aggregates showed a problem of nonspecific a11 antibody binding. It was subsequently discovered (by mass spectrometry of urine protein samples) that urine protein aggregates in the samples comprise both heavy and light chains of misfolded human immunoglobulin (IgG), as it is non-specifically recognized (e.g., cross-reacted with) by most secondary antibodies. After screening the specificity of a large number of secondary antibodies, it was found that the use of pre-adsorbed secondary antibodies with IgG is critical to ensure specificity.
When the secondary antibody in the experiment for detecting the urinary protein aggregate by using the A11 polyclonal antibody is pre-adsorbed by human IgG, the detection sensitivity is greatly improved because the non-specific binding of the secondary antibody to the misfolded protein and the light and heavy chain of immunoglobulin (IgG) in the urinary protein aggregate is reduced. Using this test method, whether the a11 test is positive will correlate with the severity of the symptoms. FIG. 10 is a photograph of a nitrocellulose membrane subjected to Western blotting after detection of a spot on a urine sample using three polyclonal antibodies A11, OC and Officer. The three polyclonal antibodies (A11 against a broad conformation, OC against fibrotype, Officer against some annular structures) showed immunological activity against pre-eclamptic patient samples (shown in the figure as urine, blood, cerebrospinal fluid (CSF) and placental debris; PE: pre-eclampsia; CRL: control). The Officer antibody is believed to be specific for a protein conformation that may form channels or voids with amyloid-like channels in RBC analogs, thereby possibly accounting for hemolysis in preeclamptic patients. Urine, blood, cerebrospinal fluid and placental debris were also tested. The data show that preeclampsia is a conformational abnormality characterized by amyloid silence. Simultaneous a11 and Officer staining indicated that the misfolded protein intermediate had a tendency to aggregate into a void-like structure (amyloid channels), which may be associated with clinical disease manifestations.
Figure 11 shows representative results of the urine spot test for 3 groups of women. The 3 groups are respectively: pre-eclampsia group (PE), cHTN, and control. Each spot contained 40mg of urine protein. The arrow marks the position of the sample point. The film was compared to the sample using a frame, the position of the sample being marked by a black circle. The results indicate that urine samples from many women with preeclampsia and a positive result on congo red staining have a11 immunoreactivity, although not all congo red positive women. This data indicates that a11 positive correlates with the severity of the symptoms.
Urine from women with severe preeclampsia showed an increase in A11 and Officer immune activity (sPE: 42 + -8, cHTN: 9 + -6, CRL: 3 + -1U/. mu.g, P <0.001), but not OC, which was independent of GA values. The intensity of urine a11 and Officer spot test results correlated with the severity of hypertension (P0.007) and proteinuria (P0.005).
In further experiments, it was examined which proteins in PE urine were involved in the formation of aggregates recognized by a 11. Two high molecular weight, A11 positive bands were excised from the non-reducing SDS PAGE gel. Identification of protein soluble oligomeric chains in sPE urine was performed using trypsin digestion followed by feeding the samples into a Keck instrument. The following identification results were verified by mass spectrometry: heavy and light chains of immunoglobulins, ceruloplasmin and interferon inducible protein 6-16 protein (GIP3, IFI6-16), where the interferon inducible protein was found to interact with Alzheimer's disease presenilin-2 protein, modulating apoptosis. The presence of IFI6-16 was verified by its antibodies in the preeclamptic urine spot test. This antibody was a mouse polyclonal antibody from Novus Biologicals Inc, Littleton, co, No. H00002537-a 01.
The above findings, that proteins in urine from preeclamptic patients have the immunological activity of antibody A11, including heavy and light immunoglobulin chains, ceruloplasmin and IP 6-16 proteins (GIP1, IFI6-16), demonstrate that quantification of one or more such protein aggregates in placenta and/or urine can also be used in the diagnosis and prognosis of preeclampsia.
Example 4 sample evaluation
Methods for preparing antigens and antibodies according to the Kaied et al article "fibrous specific transformation of monoclonal antibodies from antigenic peptides and fibrous oligomers present in recombinant oligomers" Molecular Nuoerderization 2007, 2:18, described herein as:
the preparation method of the fiber antigen comprises the following steps: 2mg/ml of A.beta.42 peptide was stirred in 50% HFIP/H2O, 0.02% sodium azide for 7 days. Subsequently, HFIP was evaporated in a stream of nitrogen and the sample was stirred for 7 days and dialyzed against PBS (molecular weight cut-off 10,000 Da). The fibers obtained were examined by EM and the purity thereof was checked by using an antibody against the oligomeric protein, based on the absence of the oligomeric protein. The antigen was used to immunize two new zealand white rabbits (Pacific Immunology Corp., Ramona, CA, 92065) using IACUC certified methods. Each rabbit was immunized with 500. mu.l of antigen in Complete Freund's Adjuvant (CFA) followed by boosting with 500. mu.l of antigen in Incomplete Freund's Adjuvant (IFA) every 4 weeks for a total of two boosts.
Preparation of fibers and oligomeric chains: a β fibers and fiber oligomers were prepared by dissolving 0.3mg of lyophilized A β 42 in 150 μ l of Hexachloroisopropanol (HFIP) for 10-20 minutes at room temperature. The obtained Abeta solution is added into DD water to prepare into a concentration of 80 mu M, and the solution is placed into an organic silicon centrifuge tube. After incubation at room temperature for 10-20 minutes, the samples were centrifuged for 15 minutes, and the supernatant fraction (pH 2.8-3.5) was transferred to a fresh silicone tube using 14000 XG rpm and placed in a weak stream of nitrogen for 10 minutes to evaporate the HFIP. After that, the sample was stirred using a Teflon coated micro stirring bar at 22 ℃ for 24 hours at 500 RPM. This method was originally intended to produce A11-positive fibrillar precursor oligomers, but more recent studies have shown that this method can also produce OC-positive fibrillar oligomers (Kayed et al, "Common Structure of soluble amyloid oligomers Common molecular biology of pathogenesis", Science2003, 300 (5618): 486-. The fibers and fiber Abeta 42 oligomers were separated by centrifugation at 100000 XG for one hour at 4 ℃. The supernatant containing the fibrillar oligomers and the precipitate containing the fibrils are separated and collected. The pellet was resuspended in an equal amount of water. Alternatively, 1mg of lyophilized Abeta 42 was dissolved in 200. mu.l of DMSO and allowed to stand at room temperature for 10 to 15 minutes to form a fiber oligomer. Fiber oligomers in DMSO were separated by a TSK-GEL SuperSW2000 column (Tosoh Bioscience LLC) using 10mM phosphoric acid at pH 7.4, according to their size, setting a flow rate of 0.3 ml/min.
As a result, the
Since the a11 antibody is not specific for oligomeric forms of the protein, further confirmation using a monoclonal antibody will provide more useful information. Thus, urine samples of A11 positive and negative were tested using a panel of mAbs that were induced by the same antigen as the A11 antibody (Science 300: 486-489, 2003), but which have different affinities and tendencies for different types of fibrin oligomer conformations than A11. We obtained 10 test strips of urine samples from women with pre-eclampsia (PE) or control group (CRL) women. Fig. 12 shows the a11 immunoreactivity results obtained in yale laboratories (lane 1). Band 2 used the same reagents and methods except that the a11 antibody was deleted and used as a control for non-specific binding of secondary antibodies. The remaining strips were sent to the UC Irvine laboratory.
FIG. 13 is a photograph of a spot of a urine sample on a nitrocellulose membrane after Western blotting (PE: preeclampsia; CRL control). Monoclonal antibodies (M204, M205, M118, M09, M55) and polyclonal antibody a11(a11), respectively, were used in these samples, as well as a negative control (Neg.) without the primary antibody, to compare non-specific binding of the secondary antibody. This experiment provided independent confirmation that the a11 antibody is immunologically active in pre-eclamptic urine and indicates that some of the more selective monoclonal antibodies are also immunologically active. These findings indicate that monoclonal antibodies recognizing different oligomer conformations of the pre-fibrin, such as monoclonal # M204, # M205 and # M89, can be used for the diagnosis of pre-eclampsia.
Urine samples taken from women with preeclampsia were strongly stained by monoclonal antibody M204 and slightly stained by M205, but this was not the case for the control group. Some of the preeclampsia samples were also stained with the M89 antibody that recognizes the ring fibril. Fig. 13 shows that the PE pathology stained most deeply by M89 also had early clinical regression consistent with HELLP (#4 cases). In contrast, #6 cases (negative for M89, but positive for M204, M205 and A11) were diagnosed with severe preeclampsia based on a single criterion of lung edema only, whereas other PE women were mostly diagnosed by blood pressure or urine protein. Case # 6 subsequently develops perinatal cardiomyopathy and thus requires strong circulatory support in the intensive care unit. Differences in staining for M89 suggest that case # 6 may have a unique etiology and may be able to differentiate these diseases by differential immunological activity of conformational monoclonal antibodies, such as monoclonal antibodies 09 and 89 against the ring fibrils.
EXAMPLE 5 preparation of antibodies
Two new zealand white rabbits were injected with antigen containing Α β 1-40 linked to gold particle colloid via a carboxyl terminal thiol ester at the terminal thiol. One hundred micrograms of polypeptide polymeric antigen was injected with incomplete freund's adjuvant and booster injections were performed every 3 weeks using the same antigen for about 5 months. After the immune response was confirmed to be equivalent to that of the a11 antibody, one animal was sacrificed, its spleen was harvested and lymphocytes therein were used to prepare hybridoma cells by standard methods.
After culturing the hybridoma cells for a sufficient period of time, the supernatant from the polyclonal well is removed and screened for antibodies specific for conformation-dependent fibrillar precursor oligomers, here using a β fibrillar precursor oligomers as the initial screening reagent. A β monomers, fiber precursor oligomers, and fibers were used in a second round of screening to remove antibodies that were not conformation dependent and interacted with all the various a β conformations. Approximately 118 polyclonal wells were selected for having an immunological activity exceeding the standard value of 0.5 AU. These polyclonals are further classified and the resulting monoclonals are further screened and characterized by Α β monomers, fiber precursor oligomers and fibers.
After the second round of selection, clones with unique tendencies toward fiber precursor oligomers or fibers are selected. Representative clones were selected by comparison with the a11 polyclonal antibody and 6E10, a sequence-specific murine monoclonal antibody. The antibodies were tested for conformation specificity and sequence specificity by dot blot. The dot blot assay included 1. mu. g A β monomer, fiber precursor oligo and fiber, as well as 1. mu.g α synuclein fiber precursor oligo, immunoglobulin light chain, prion 106-126 polypeptide fragment, KK (Q40) KK and calcitonin. The a11 polyclonal antibody acted with all types of celloprecursor oligomers, but not with a β monomers and fibers. 6E10 only identified samples containing A β. Clones 118, 201, 204, 205, and 206 were specific for fiber precursor oligomers, but did not recognize monomers or fibers. This set of clones showed unique specificity for other identified fiber precursor oligomers. Clone 121 specifically recognized a β fibers, but did not recognize either the fiber precursor oligomers or the a β monomers.
Many clones secrete antibodies with the same specificity. The most abundant type among these was similar to clone 201, i.e., only a β fibril precursor oligomers were recognized, and no other type of oligomers were recognized. All these clones were also IgM at the same time. Clone 118 was also indistinguishable from clones 48 and 55 (data not shown)
Sequence information for two monoclonal iggs, 118 and 204, is listed below. The amino acid sequences of the diversity regions are different, consistent with their different specificities.
#118 kappa VI+Cl
AQAAEL VMTQTPASVSAA VGGTVTINCQSSESVYNSRLSWFQQKPGQPPKLLIYFASTLASGVSSRFSGSGSGTEFTLTISGVQCDDAA TYYCAGHFSNSVYTFGGGTEVWTGDPV APTVLIFPPSADLVATGTVTIVCV ANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC*(SEQ ID NO.7)
#118 Vh+Clh
AAQP AMAQSVEESGGRL VTPGTPLTLTCTVSGFSLSA YEVSWVRQAPGKGLEWIGIIY ANGNTVY ASW AKGRFTISKTSTKVDLRIPSPTTEDTATYFCARDIYTTTTNLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTHSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKTSC(SEQ ID NO.8)
#204 kappa VI+Cll
AQAAELDMTQTPASVSEPVGGTVTIKCQASQSISSYLA WYQQKPGQRPRLLIYETSTLASGVPSRFKGSGSGTEFTLTISDLECADAA TYYCQSTYENPTYVSFGGGTEVGVKGDPV APTVLIFPPSADLV ATGTVTIVCV ANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC*(SEQ ID NO.9)
#204 Vh+Clh
AAQPAMAQSVKESGGRLVTPGTPLTLACTVSGFSLNTYSMFWVRQAPGKGLQWIGIISNFGVIYY ATW AKGRFTISKTSTTVDLKITSPTTEDTA TYFCVRKYGSEWGGDLWGPGTL VTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTL TNGVRTFPSVRQSSGL YSLSSVVSVTSSSQPVTCNV AHPATNTKVDKTVAPSTCSKTSC(SEQ ID NO.10)
#205 kappa VI+Cll
AQAAEL VMTQTPSSVSAA VGGTVTISCQSSESVYNNNYLSWYQQKPGQPPKRLIDSASTLDSGVPSRFKGSGSGAQFTLTISDLECDDAA TYYCAGA YVNWMRIFGGGTEVVVKGDPVAPTVLIFPPSADLVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC*(SEQ ID NO.11)
#205 Vh+Clh
MAQSVEESGGRLVTPGTPLTLTCTASGFSLINYYMNWVRQAPGKGLEWIGLITGW ADTYY ANSAKGRFTISKTSSTTVDLKITSPTTDDTATYFCVRGGHTNIISLWGPGTLVTVSSGQPKAPSVFPLPPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRIFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKTSC(SEQ ID NO.12)
Monoclonal antibodies against Α β oligomers and fibers:
although polyclonal antibodies have strong specificity for the immune response of different aggregated forms of the amyloidogenic protein, monoclonal antibodies have distinct advantages in determining fine structural changes in the amyloidogenic aggregates, identifying the structural and pathological importance of the aggregates. We initially focused on antibodies directed against the fiber precursor amyloid cluster, but immunized mice with gold colloid-conjugated a β as we prepared a 11. We tried to prepare mouse monoclonal antibodies at two different suppliers by using 4 different strains of mice. Although we obtained good titers of conformation-dependent IgG in mouse polyclonal plasma, we were still able to clone only IgM-secreting hybridoma cells from mouse spleen cell fusion products. We tried a different route of immunization, with 6 months of immunization injections, and using lymphocytes of no origin (peripheral and lymph nodes), but we have not been able to obtain clones other than IgM. The reason why IgG-secreting clones could not be obtained from mouse hybridoma cells is still unknown. Although these IgM clones have some utility, they are not suitable under many application conditions, and we have signed a contract with Epitomics, Inc to make rabbit monoclonal IgG. Although we used the same antigen and screening method as when obtaining mouse monoclonal antibodies, we obtained more positive independent clones (>200), many of which were IgG clones (data not shown). Many clones in between have similar phenotypes and are grouped into 6 different groups we have currently identified. (FIG. 1)
The specificity of the monoclonal antibodies we obtained is of interest, mainly for several reasons (FIG. 1). Although we screened antibodies against A β monomers, oligomers and fibers, all monoclonal antibodies obtained by injection of A11A β C-terminal mercaptoester linked gold colloid were conformationally specific. None of the clones recognized a similar 6E10 monomer. This indicates that the immune response to the solid-state antigen is highly conformationally specific. No antibody could recognize both pure fiber and pure oligomer samples, indicating that the distribution of these epitopes is mutually exclusive.Second oneMost antibodies (M118, M204, M205, M206) recognize "universal epitopes" distributed among the fiber precursor oligomers derived from other protein and peptide chain sequences. However, in this group of antibodies recognizing a "universal" fiber precursor oligomer epitope, there is considerable variation in the oligomer type recognized by the antibody. All universal monoclonal antibodies recognized the a β oligomers as they were used for the first screening, but each antibody had a more stringent specificity than the a11 polyclonal immune response. For example, M204 can strongly recognize most oligomers, but reacts significantly less strongly with immunoglobulin light chain oligomers. M205 reacted strongly with alpha synuclein and light chain oligomers, but Is unreactive with the Raney virus 106-126, polymeric Q and calcitonin oligomer. M118 favors light chains and polymeric Q, but has no effect on synuclein, prion, or calcitonin oligomers. These results show that the broadly distributed celloprecursor oligomers in this group carry a variety of different antigenic determinants, whereas monoclonal antibodies can recognize these unique antigenic determinants.Third stepSome monoclonal antibodies are both conformation dependent and sequence specific. M201 recognizes only a β oligomers, while M121 recognizes only a β fibers. M118, M204 and M205 are IgG and the other antibodies are IgM.
Example 6
Monoclonal antibodies were prepared by Epitomics, Inc in rabbits. New zealand white rabbits were injected with a β ring fibrils, prepared using the method described by Kayed et al in j.biol.chem.2009 (1). The purified a β ring fibrils are prepared starting from a β fibril precursor oligomers prepared as described in (2) above. The APF was prepared by adding 5% (v/v) n-hexane to the fiber precursor oligomer solution, and the samples were mixed by vortex shaker for 1 minute every 5 minutes for a total of 50 minutes. Subsequently, the samples were dialyzed in water using a dialysis membrane with a MW cut-off of 10 kDa. Rabbits were injected 7 times in total, with 500 μ g of a β ring fibrils every 3 weeks apart. The plasma was screened for the specific titer of the ring fibrils, and the rabbit with the highest titer was selected for monoclonal antibody production. The resulting supernatant of hybridoma cells was first screened by ELISA for a β ring fibrils, a β fibers and a β monomers. Wells with optical densities above 1.0 for a β ring fibrils, and background levels of activity for a β fibers and a β monomers were selected as secondary screens. Approximately 100 wells were selected for secondary screening, screening for a β ring fibrils (APFs), α hemolysin pores, a β fibers, a β fiber precursor oligomers, a β monomers, and a β in 0.1% SDS solution, using dot blot hybridization. The results are shown below.
In the secondary screening, monoclonal 09 and 89 were immunoreactive only to the alpha hemolysin pores. We also identified their immune responses to α hemolysin and a β dissolved in 10mM NaOH, and the results of western blotting are shown below.
Both monoclonal antibodies 09 and 87 were found to be able to react with the approximately 65kDa band in the Western blot. M87 also reacted with a band of approximately 40 kDa. The alpha hemolysin band at 65kDa also reacted significantly with the rabbit polyclonal plasma used to prepare the monoclonal antibody.
1.Kayed,R.,A.Pensalfmi,L.Margol,Y.Sokolov,F.Sarsoza,E.Head,J.Hall,and C.Glabe.2009.Annular proto fibrils are a structurally and functionally distinct type of amyloid oligomer.J Bioi Chern 284:4230-4237.
2.Kayed,R.,and C.G.Glabe.2006.Conformation-dependent anti-amyloid oligomer antibodies.Methods EnzyrnoI413:326-344.
The use of this invention requires conventional cell biology, cell culture, molecular biology, microbiology, DNA hybridization, and immunology techniques that are conventional in the art, unless otherwise indicated. These techniques are all described in the literature.
The invention is not limited in its application to the details of construction and the arrangement of components set forth in the preceding text or drawings. This invention can be used in other cases as well, and is applied by various methods. The phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of the terms "comprising," "including," or "having," "containing," "involving," and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.
Claims (9)
1. Use of a labelled reagent for misfolded protein aggregates for the preparation of an in vitro diagnostic reagent for the in vitro diagnosis of pre-eclampsia or for predicting the likelihood of future development of pre-eclampsia in an individual.
2. The use of claim 1, wherein the diagnosis or prognosis is further assessed by performing one or more additional tests selected from the group consisting of: blood pressure measurements, edema assessment, abdominal pain, headache development and visual problems.
3. The use of claim 1, wherein the diagnosis or prognosis is further assessed by detecting preeclampsia-related biomarkers selected from the group consisting of: sFlt-1, PIGF, VEGF, albumin, SerpinA1, IFI6 and ceruloplasmin.
4. The use of any one of claims 1-3, wherein said labeling agent for misfolded protein aggregates is a dye that labels said misfolded protein aggregates or a conformation-specific labeling agent for said misfolded protein aggregates.
5. The use of claim 4, wherein said labeling agent for misfolded protein aggregates is Congo red, or said conformation-specific labeling agent is a conformation-dependent and amino acid sequence-independent antibody.
6. A kit for in vitro diagnosis of preeclampsia or prediction of the likelihood of future development of preeclampsia in an individual, comprising:
a labeling agent that misfolds protein aggregates; and
and (4) positive control.
7. The kit of claim 6, further comprising a support surface having an affinity for proteins.
8. The kit of claim 7, further comprising a wash solution of said labeling reagent for removing unbound misfolded protein aggregates.
9. Use of an inhibitor of misfolded protein aggregates for the manufacture of a medicament for attenuating the symptoms of or treating preeclampsia in a pregnant woman.
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CN110208540B (en) | 2024-02-09 |
CN110187121A (en) | 2019-08-30 |
HK1250392A1 (en) | 2018-12-14 |
US20210255192A1 (en) | 2021-08-19 |
US11125758B2 (en) | 2021-09-21 |
CN110208540A (en) | 2019-09-06 |
CN104777307B (en) | 2019-10-18 |
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